F.W. van Leeuwen

Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat.

SummaryPerfusion of rat brain followed by immersion fixation with 2.5% glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.

Vasopressin cells in the bed nucleus of the stria terminalis of the rat: sex differences and the influence of androgens

A sex difference in the number of vasopressin-immunoreactive cells was found in the bed nucleus of the stria terminalis of the rat. The number of cells found in males exceeded the female corresponding value. A sharp decrease in the number of vasopressin-immunoreactive cells was noted 21 weeks after the castration of adult male rats. This decline could be reversed completely by a 5-week testosterone substitution therapy.

Can specificity ever be proved in immunocytochemical staining.

Any investigator working with immunocytochemical (ICC) localization techniques can appreciate the statement of Petrusz et al. (1) that specificity is the most difficult criterion to fulfill. According to these authors, the only direct way to establish specificity is absorption of the antibody by an antigen of high purity. Ever since we compared systematically the ICC localization data in tissue with those obtained in model systems we have, however, noticed that the absorption technique alone is absolutely insufficient to prove the specificity for the homologous antigen. Since overestimation of the value of the absorption test used in this way is found throughout the ICC localization literature, we thought it worthwhile to react and present some data that illustrate our point of view, and to suggest possible alternative specificity tests. We agree with Petrusz et al. (1) that radioimmunoassay (RIA) cannot help us to characterize the antibodies for ICC procedures. The difference between the specificity revealed by these two techniques may be at least partly due to the much higher dilution in which the antibodies are used in the RIA as was illustrated by immunoelectronmicroscopical data showing dilution dependent specificity (5). Specificity has thus to be studied in the same antibody dilution as is used for the ICC localization technique. The dissimilarity between RIA and ICC holds not only true for the specificity but also for the potency of the antiserum. During the course of antibody development against different hormones in rabbits, we frequently observed that the titre in the RIA system was rising or remained elevated (e.g., 1:10”) while the fluorescence obtained with the same antibody in tissue or on model systems gradually decreased to useless values (cf. e.g., 4). As Petrusz et al. (1) already stated, the proof that positive staining results exclusively from immunochemical binding of the antibodies in the primary antiserum to the tissue presents relatively little difficulty. Although the increasing dilution of the primary antiserum as proposed by these authors may be of some value, possible disturbing factors causing nonimmunochemical reactions will also be diluted in this way. It seems, therefore, preferable to test the pre-immune serum of the same rabbit in the same dilution to prove the (non)immunocytochemical nature ofthe reaction. Notjust the plasma from a single rabbit since each animal may have its own characteristics in this respect. For the same reason it seems even more advisable to use plasma that has been absorbed to the antigen as a check for the antibody antigen reaction. This plasma contains all the factors that influence the ICC reaction with the exception of the antibodies that will be studied further. We disagree fundamentally with Petrusz et al. (1) with respect to the value of the absorption test alone to prove specificity. If no ICC staining is obtained anymore after absorption of the antibody by the antigen, it proves no more than that all the antibodies were bound to the added antigen. In the first place it does not exclude staining caused by unwanted or unexpected antibodies which were raised because of impurities in the injected antigen which is also used for the absorption test. Most antigens have to be purified from biological material and will thus never be absolutely pure. In the second place it does not exclude cross reaction. This phenomenon is even not mentioned by Petrusz et al. (1) although it is one of the main problems of the ICC localization techniques, even when, theoretically speaking, pure antigen could be used. The data we obtained with antibodies that were raised to synthetic argininevasopressin (AVP) can serve as an illustration of this point. These antibodies showed a good positive reaction in rat brain and pituitary sections. However, in homozygous Brattleboro rats that lack AVP by a genetical defect, a bright immunofluorescence was also found using these antibodies. This fluorescence appeared to be due to cross reaction with the structurally closely related hormone oxytocin. Yet, solid phase absorption to AVP of this antibody against AVP prevented any ICC staining and would thus be “specific” according to Petrusz’ criteria. Specific localization of AVP was only possible after removal of the cross reacting antibodies by solid phase

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.

The localization of oxytocin, vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis.

SummaryThe hypothalamic hormones arginine-vasopressin (AVP), oxytocin (OXT), somatostatin (SOM), and luteinizing hormone-releasing hormone (LHRH) were localized in the rat neurohypophysis by the use of semithin serial sections and the unlabeled antibody enzyme method. Clusters of AVP fibres are present within the central region of the neural lobe, clusters of OXT fibres mainly in the peripheral part. The AVP fibres enter bilaterally into the neural lobe.The results call into question previous reports on the presence of AVP on receptors in the pars intermedia cells, since incubation with anti-AVP resulted in similar staining in the pars intermedia of the Wistar and homozygous Brattleboro rat, a mutant strain deficient in AVP. The same intermediate lobe cells are stained after incubation of serial sections with anti-AVP and anti-α-melanocyte-stimulating hormone (α-MSH). This staining of anti-AVP could be removed by solid phase absorption to α-MSH and is thus most probably due to cross reaction with α-MSH. SOM fibres appear to be present in the peripheral parts of the proximal neurohypophysial stalk and mainly lateral in its more distal parts. In the neural lobe they rapidly decrease in number, although some fibres continue into the distal part of the neural lobe, running bilaterally and situated adjacent to the pars intermedia. The SOM staining within magnocellular elements, which has been reported in the literature, can most probably be explained by cross reaction of anti-SOM with neurophysins. LHRH fibres are very scarce in the neurohypophysial stalk and absent in the neural lobe.

Sex Differences in Vasopressin and Other Neurotransmitter Systems in the Brain

Publisher Summary The sex differences that are found in neuronal connectivity suggest that such differences may also be expected with respect to the neurotransmitter content measured within sexually dimorphic areas. This chapter discusses the various aspects of the study of sex differences in neurotransmitter systems, and the vasopressin innervation of the brain is discussed as an example of the use of immunocytochemistry in studying sex differences. Several methods have been developed for studying the anatomy of neurotransmitter systems: (i) the histofluorescent demonstration of the indolamines and catecholamines, (ii) enzymes histochemical procedures for cholinergic neurotransmitter pathways, and (iii) immunocytochemistry, which is potentially the most versatile method, because it does not depend on the detailed knowledge of either the chemical nature or the metabolizing enzymes for a given neurotransmitter. Studies of the vasopressinergic innervation of the brain have demonstrated that immunocytochemistry in conjunction with tracing and lesion studies forms a powerful tool to study the anatomical background of a particular sex difference. However, one major problem of immunocytochemical methods is that it is difficult to raise an antiserum, which is directed specifically against a single antigen, which makes it necessary to extensively test all sera and, if necessary, to purify them. An advantage of the application of immunocytochemistry is that it can readily be combined with other techniques, for example, with steroid receptor labeling. The study of the neurotransmitter content of the various components of a given sexually dimorphic system can also help to elucidate its functional significance, if only because the knowledge about the neurotransmitters involved makes it possible to use pharmacological tools, such as local administration of specific agonists or antagonists.

Coexistence of vasopressin, neurophysin and noradrenaline immunoreactivity in medium-sized cells of the locus coeruleus and subcoeruleus in the rat

Vasopressin-and neurophysin-immunoreactive cells have recently been demonstrated in the rat locus coeruleus (A6) and subcoeruleus (A7). Using consecutive 5 microns thick frozen sections, medium-sized cells throughout the locus coeruleus area, but predominantly in the posterior parts of the A6 displayed coexistence for vasopressin and noradrenaline or neurophysin and noradrenaline immunoreactivity. The putative projection areas of putative fibers from vasopressin-containing cells in the locus coeruleus still remain to be elucidated.

Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat

SummaryAn immunoelectronmicroscopic method for the specific localization of neurohypophyseal hormones was developed in neurohypophyses of Wistar and Brattleboro rats, the latter strain being homozygous for diabetes insipidus. If the proper precautions were omitted, a marked cross reactivity between antivasopressin and antioxytocin preparations was found. Cross reaction of an antivasopressin plasma with oxytocin, at a dilution of less than 1∶1600, resulted in electron density of all granules within neurosecretory fibres of the Brattleboro and Wistar neurohypophyses. However, this cross reactivity could be eliminated either by sufficient dilution of the antiplasma, or by its purification. Purification of the antibodies was performed by absorption to agarose beads coated with the cross reacting component. Upon incubation with antivasopressin (diluted unpurified 1∶1600 or purified 1∶80) and unpurified antioxytocin (1∶400) plasma, sections of a Wistar neurohypophysis revealed two types of neurosecretory fibres, containing either electron dense or lucent granules. Oxytocin and vasopressin containing neurosecretory fibres were found as clusters in the neurohypophysis. The specificity of both unpurified antivasopressin (1∶1600) and antioxytocin (1∶400) plasma was confirmed on serial sections of a Wistar neurohypophysis, alternately incubated with the solutions of the two antibodies.These data prove that the one-cell-one-hormone hypothesis holds true for the hypothalamic-neurohypophyseal system.

Neuron-glia interactions in the release of oxytocin and vasopressin from the rat neural lobe: The role of opioids, other neuropeptides and their receptors

The release of the neurohormones oxytocin and vasopressin from the neural lobe into the circulation is regulated in a complex manner, which has only been partly elucidated. At the level of the neural lobe, regulation of release can occur by various endogenous compounds that act on specific receptors present on the nerve terminals themselves. In addition, release may be modulated by an alternative pathway in which the local glia cells, the pituicytes, are involved. It is especially the latter pathway that is discussed in detail in this commentary.

Immunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus

SummaryThe classical areas for arginine-vasopressin (AVP) synthesis are the magnocellular supraoptic (SON) and paraventricular nuclei. More recently AVP was also demonstrated in neurons of the parvocellular suprachiasmatic nucleus (SCN) of the rat. This result was substantiated in the present study by means of immunoelectron microscopy, by subjecting sections to antivasopressin plasma. Conventional electron microscopy revealed dense-core vesicles in the SCN cell bodies and fibres (mean diameter 94.7±0.9 nm and 84.0±1.1 nm respectively). These vesicles were infrequent within the cell bodies and could not be accumulated by ethanol administration. Immunoelectron microscopy showed a positive reaction in the cell bodies and fibres within vesicles of 93.7±1.1 nm and 98.5±1.2 nm respectively. By comparison, the cell bodies and fibres of the SON showed immunoreactive granules of 143.0±1.8 and 147.3±1.8 nm respectively. The presence in the SCN of AVP in vesicles of different size than those in the SON suggests that synthesis of this substance is indeed occurring within the SCN cells.