Jennifer H. Steel

Increased Hypothalamic Neuropeptide Y Concentrations in Diabetic Rat

Central and lateral hypothalamic concentrations of 10 regulatory peptides were measured by radioimmunoassay in streptozocin-induced diabetic (STZ-D) and matched control rats between 1 day and 14 wk after diabetes induction. After 2 wk, both central and lateral hypothalamic neuropeptide Y (NPY) concentrations in STZ-D rats were consistently higher than those found in control rats, with significant 30–50% increases at 4 wk in the central hypothalamus, and at 6 and 14 wk in both central and lateral hypothalamus. Immunocytochemical studies in 4- and 6-wk STZ-D animals showed the appearance of intensely NPY-positive swollen cell bodies in the supraoptic nucleus and a subjective increase in NPY staining of medial hypothalamic nerve fibers. Central hypothalamic concentrations of three other peptides were significantly greater in STZ-D animals than those in control animals at single points (neurotensin, 1 day; calcitonin gene-related peptide, 2 wk; neurokinin, 4 wk). Hypothalamic concentrations of the other six peptides examined (bombesin, galanin, neuromedin B, substance P, somatostatin, and vasoactive intestinal peptide) did not differ significantly between STZ-D and control groups at any time. However, galanin immunostaining in the supraoptic and magnocellular paraventricular nuclei was strikingly concentrated in a reduced number of distended cell bodies. Hypothalamic peptide changes in STZ-D could be related to metabolic disturbance, changes in energy and water balance, altered pituitary function, or other factors. Persistently elevated concentrations of NPY, a very potent central stimulant of eating and drinking, may mediate the hyperphagia and polydipsia characteristic of STZ-D.

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

SummaryThe identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-μm serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.

Observer variation in quantification of immunocytochemistry by image analysis.

SummaryThis paper reports the findings of a study designed to examine observer variation as a source of inaccuracy inherent in the use of computer-assisted image analysis to measure areas of stained tissue. The rat pituitary immunostained for prolactin and galanin was used as an example to estimate patterns of immunoreactivity exhibited by different cell types. Six observers, with differing experience, selected grey level threshold values on 40 fields of images of stained tissue making three repeats of each field. The 40 fields consisted of 20 serial pairs of colocalized fields, one immunostained for prolactin, the other for galanin. The 20 pairs consisted of four pairs from each of five animals.Analysis of observer variation in the selection of threshold values showed large differences in the within-and between-observer variation. Analysis of the components of variance in the estimation of the ratios of stained tissues showed that the major source of variation was the within-observer component. An additional experiment using two observers, where half of the images were compared to the original microscope images before setting threshold levels, showed that the opportunity to make a comparison did not reduce observer variation.It is suggested that any study which uses semi-automatic methods to segment regions of a digital image can benefit from an analysis of this kind so that the sources of variation can be determined to enable maximum discriminating power in future studies.

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats

SummaryWe have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.

Peptidylglycine α-amidating monooxygenase (PAM) immunoreactivity and messenger RNA in human pituitary and increased expression in pituitary tumours

Bioactivity of many peptides depends upon post-translational α-amidation of inactive precursors by two enzyme activities known collectively as peptidylglycine α-amidating monooxygenase (PAM). PAM enzymes are particularly abundant in the pituitary. The distribution of PAM immunoreactivity and messenger ribonucleic acid (mRNA) in the adult human pituitary and in pituitary tumours was investigated by use of immunocytochemistry and in situ hybridisation. Immunoreactivity was present in numerous cells of the anterior lobe: staining was intense in a proportion of gonadotrophs and folliculo-stellate cells, but weaker in the majority of somatotrophs and lactotrophs, a few corticotrophs and occasional thyrotrophs. PAM staining was also present in nerves, pituicytes and some endocrine cells within the posterior lobe (the human intermediate zone). Forty pituitary tumours of various types were immunoreactive for PAM; more intensely and uniformly stained than normal anterior lobe. In situ hybridisation with digoxigenin-labelled probes demonstrated intense labelling for PAM mRNA in numerous cells in normal anterior pituitary and in tumours. Many regulatory peptides that require amidation for activity, potential targets for PAM, are present in the pituitary. Many tumour growth factors also require amidation and PAM may regulate these mitogenic peptides in tumours.

Advantages of In situ Hybridisation over Direct or Indirect In situ reverse Transcriptase–polymerase Chain Reaction for Localisation of galanin MRNA Expression in Rat Small Intestine and Pituitary

In situ hybridisation (ISH) and direct or indirect in situ reverse transcriptase–polymerase chain reaction (RT–PCR) were used to detect galanin mRNA in paraffin sections of rat intestine and pituitary. With conventional ISH, a subset of intestinal neuronal ganglion cells and anterior pituitary endocrine cells were labelled. Direct in situ RT–PCR also labelled some cells in pituitary but not in intestine. Negative controls were unlabelled, but sections with 3′ primer alone for RT–PCR appeared positive. No signal was apparent using the indirect in situ RT–PCR method. Investigation of the specificity of solution phase RT–PCR using RNA extracts from pituitary or intestine revealed that additional PCR products were detected under some conditions. The sequences of these PCR products suggested that one was the result of mispriming and single primer PCR, which could also have occurred in situ. Alternative galanin primers gave only the predicted RT–PCR product in solution phase yet still gave artefacts in tissue sections using direct in situ RT–PCR. ISH with probes transcribed from the correct PCR product gave identical labelling to the original galanin riboprobe. In conclusion, direct in situ RT–PCR is unreliable and requires validation, while indirect in situ RT–PCR may fail even though sufficient target exists for detection with conventional sensitive riboprobe ISH.

Combined use of immunocytochemistry and in situ hybridization to study β thyroid-stimulating hormone gene expression in pituitaries of hypothyroid rats

Immunocytochemistry and in situ hybridization were used to demonstrate beta thyroid-stimulating hormone (TSH) immunoreactivity and beta TSH messenger RNA (mRNA) in the same tissue section of rat pituitary. Sections (10 micronsP of 4% paraformaldehyde-fixed pituitaries from surgically thyroidectomized and intact male rats were first hybridized with a 32P-labelled complementary RNA beta TSH probe and were then immunostained for beta TSH. In both sets of animals beta TSH mRNA and beta TSH immunoreactivity were simultaneously localized to many thyrotrophs, although there was considerable heterogeneity in mRNA labelling intensity between individual cells. In hypothyroid rats more cells were positive with both probe and antiserum, and the intensity of mRNA labelling in most quantified by direct apposition of hybridized sections to autoradiography film, and the image grey levels were measured using an image analyser to convert them into equivalent amounts of radioactivity. In hypothyroid rats this amount was 22 times greater than in controls (control 4.77 +/- 0.84 attomoles mm-2; hypothyroid 107.06 +/- 14.2 attomoles mm-2; mean +/- SEM p less than 0.001). In situ hybridization has been used in combination with immunocytochemistry, to demonstrate beta TSH and mRNA and immunoreactivity at a cellular level in rats with different thyroid status.

Galanin: Distribution, ontogeny and expression following manipulation of the endocrine and nervous systems

Since its discovery in extracts of pig intestine (Tatemoto et al., 1983), galanin has been found in many other locations in the central and peripheral nervous systems and also the anterior pituitary gland (Rokaeus et al., 1984; Skofitsch and Jacobowitz, 1985a; Bishop et al., 1986; Kaplan et al., 1988). Like most, if not all, known regulatory peptides, it coexists with neurotransmitters and other peptides (Melander et al., 1986), and its levels vary with physiological state (Gabriel et al., 1990). No definite roles have yet been ascribed to the peptide, though it seems to be involved in release of various hormones such as growth hormone and prolactin (Bauer et al., 1986; Ottlecz et al., 1986; Murakami et al., 1987; Melander et al., 1987) and may act as a growth promoter (Woll and Rozengurt, 1989). Experimental work has been confined to the rat and little is known of the distribution and effects of galanin in man. In this chapter we describe the immunocytochemical distribution of galanin in adult human brain and in developing and adult human and rat spinal cord and dorsal root ganglia. Changes in galanin immunoreactivity in the rat after experimental nerve injury and in man in some pathological conditions are described. Galanin messenger RNA (mRNA) has been shown in some of the material, Anterior pituitary galanin-like immunoreactivity in the rat has been assessed during development and after manipulation of the endocrine system (oestrogen treatment, gonadectomy) and human pituitary galanin has been examined in autopsy material and in tumours.

Changes in Prolactin and Proopiomelanocortin Messenger RNA in Rat Pituitary as Shown by in Situ Hybridization

Although the peptide content of endocrine cells is reliably demonstrated by immunocytochemistry, it may be unrelated to the immediate physiological state of the cell and the turnover rate of cellular products. By studying the mRNA present in the cytoplasm by in situ hybridization, it is possible to evaluate the synthetic activity of a cell. Although the cytoplasmic mRNA content is dependent on factors such as the rate of post-transcriptional processing and mRNA stability as well as on the rate of formation of primary gene transcripts (1), in situ hybridization of mRNA is still a useful technique for comparing changes in physiological activity. Our hypothesis was that the combination of immunocytochemistry and in situ hybridization might provide a more complete picture of the dynamic endocrinology of a tissue. Prolactin and POMC gene expression in rat pituitary were used as examples. We studied prolactin synthesis and secretion during pregnancy and lactation, and also after ovariectomy, because estrogen is known to stimulate the synthesis and secretion of prolactin in vivo and in vitro (2,3), such regulation taking place at the level of prolactin gene transcription (4,5). Prolactin mRNA levels increase in pituitaries of normal rats or hypogonadal mice given estrogen treatment (6,7). The technique of in situ hybridization has already been used to demonstrate increased prolactin mRNA in sections of pituitary from rats given chronic diethylstilboestrol treatment (8) or estrogen (9).