Exploring immunoreactivity of TTF-1 and AVP in hypothalamic hamartoma.

INTRODUCTION: Hypothalamic hamartoma (HH) is a rare developmental disorder presenting with gelastic seizures or precocious puberty attributed to gonadotrophin-releasing hormone expression by the hamartoma. The histogenesis of HH is uncertain, and diagnosis of HH is difficult in small biopsies due to its close resemblance to normal hypothalamic nuclei. TTF-1 and arginine vasopressin (AVP) are associated with gonadotropin-releasing hormone release. MATERIALS AND METHODS: In this study, we explored the expression pattern of TTF-1 and AVP in HH and its utility, if any, in diagnosis. We reviewed the clinical, radiologic, and histopathological features of 23 HH diagnosed over the past decade at our Institute. RESULTS: The age at presentation ranged from 11 months to 34 years with gelastic seizures (82.6%), precocious puberty (17.4%), and developmental delay (8.7%) as presenting symptoms. On imaging, all the lesions (n = 9) involved the posterior and tuberal group of hypothalamic nuclei, while 5 cases involved the anterior hypothalamus. Anatomically, the lesions involved mammillary body, arcuate and periventricular nuclei. On histopathology, 52% cases revealed nodular arrangement of small neurocytic cells separated by glial stroma. TTF-1 and AVP immunoreactivity was absent in all the cases, whereas in normal hypothalamus, AVP was expressed in periventricular nuclei. CONCLUSION: Our results suggest that immunoexpression of TTF-1 is absent in HH, particularly in those arising from the posterior hypothalamus, and this can be used in small biopsies to distinguish from a normal hypothalamus as well as from posterior pituitary tumors.

Autoimmune central diabetes insipidus in a patient with ureaplasma urealyticum infection and review on new triggers of immune response

Diabetes insipidus is a disease in which large volumes of dilute urine (polyuria) are excreted due to vasopressin (AVP) deficiency [central diabetes insipidus (CDI)] or to AVP resistance (nephrogenic diabetes insipidus). In the majority of patients, the occurrence of CDI is related to the destruction or degeneration of neurons of the hypothalamic supraoptic and paraventricular nuclei. The most common and well recognized causes include local inflammatory or autoimmune diseases, vascular disorders, Langerhans cell histiocytosis (LCH), sarcoidosis, tumors such as germinoma/craniopharyngioma or metastases, traumatic brain injuries, intracranial surgery, and midline cerebral and cranial malformations. Here we have the opportunity to describe an unusual case of female patient who developed autoimmune CDI following ureaplasma urealyticum infection and to review the literature on this uncommon feature. Moreover, we also discussed the potential mechanisms by which ureaplasma urealyticum might favor the development of autoimmune CDI.

Autoimmune central diabetes insipidus in a patient with ureaplasma urealyticum infection and review on new triggers of immune response.

Diabetes insipidus is a disease in which large volumes of dilute urine (polyuria) are excreted due to vasopressin (AVP) deficiency [central diabetes insipidus (CDI)] or to AVP resistance (nephrogenic diabetes insipidus). In the majority of patients, the occurrence of CDI is related to the destruction or degeneration of neurons of the hypothalamic supraoptic and paraventricular nuclei. The most common and well recognized causes include local inflammatory or autoimmune diseases, vascular disorders, Langerhans cell histiocytosis (LCH), sarcoidosis, tumors such as germinoma/craniopharyngioma or metastases, traumatic brain injuries, intracranial surgery, and midline cerebral and cranial malformations. Here we have the opportunity to describe an unusual case of female patient who developed autoimmune CDI following ureaplasma urealyticum infection and to review the literature on this uncommon feature. Moreover, we also discussed the potential mechanisms by which ureaplasma urealyticum might favor the development of autoimmune CDI.

Immunohistochemical detection of NRSA on small cell lung cancer with a monoclonal antibody (MAG-1) that recognizes the carboxyl terminus of provasopressin.

A single monoclonal antibody (MAG-1) directed against the C-terminal 18-amino acid region (VAGc18) of provasopressin was examined as an agent for recognizing the tumor-specific NRSA marker common to small cell lung cancer (SCLC) in formalin-fixed tissues with ABC immunohistochemistry. SCLC tumors were obtained from several tissue locations and included primary, metastatic, and recurrent disease. Positive staining was found in 91% of cases (53/58). All five of the unreactive tumors were of the lungs or chest wall, and there did not appear to be an association of this negativity with disease stage, age, or sex. Alternatively, almost all primary lesions, almost all metastatic lesions, and all recurrent lesions examined gave a positive reaction with MAG-1. For this study, vasopressin-producing cells of the human anterior hypothalamus served as a positive control, while negative controls comprised normal lung tissue, tumor that received MAG-1 in the presence of an excess of antigen (VAGc18 peptide), or tumor reacted with a commercial IgG1 isotype as primary antibody. All of the results indicate that MAG-1 can be effectively used to selectively identify the NRSA marker on almost all SCLC tumors, at all disease stages, and at all locations. Since all four tumors tested showing no reactivity with MAG-1 gave a positive reaction for synaptophysin, it is proposed that a combined use of MAG-1 with synaptophysin antibodies could allow all SCLC tumors to be detected by ABC immunohistochemistry.

Differential cellular handling of defective arginine vasopressin (AVP) prohormones in cells expressing mutations of the AVP gene associated with autosomal dominant and recessive familial neurohypophyseal diabetes insipidus.

An unusual mutation in the arginine vasopressin (AVP) gene, predicting a P26L amino acid substitution of the AVP prohormone, is associated with autosomal recessive familial neurohypophyseal diabetes insipidus (FNDI). To investigate whether the cellular handling of the P26L prohormone differed from that of the Y21H prohormone associated with autosomal dominant inheritance of FNDI, the mutations were examined by heterologous expression in cell lines. Immunoprecipitation demonstrated retarded processing and secretion of the Y21H prohormone, whereas the secretion of the P26L prohormone seemed to be unaffected. Confocal laser scanning microscopy showed accumulation of the Y21H prohormone in the endoplasmic reticulum, whereas the P26L prohormone and/or processed products were localized in secretory granules in the cellular processes. RIA analysis showed reduced amounts of immunoreactive Y21H-AVP and P26L-AVP in the cell culture medium. Thus, the recessive mutation does not seem to affect the intracellular trafficking but rather the final processing of the prohormone. Our results provide an important negative control in support of the hypothesis that autosomal dominant inheritance of FNDI is caused by mutations in the AVP gene that alter amino acid residues important for folding and/or dimerization of the neurophysin II moiety of the AVP prohormone and subsequent transport from the endoplasmic reticulum.

[Localization of proline-rich peptide (galarmin) in brain structures of rats (immunohistochemical study)]. / Lokalizatsiia obogashchennogo prolinom peptida (galarmina) v strukturakh mozga krys (immunogistologicheskoe issledovanie).

In the present study the immunohistochemical localization of proline-rich peptide, so called galarmin, was examined in the brain structures of intact and galarmin-treated rats. Galarmin (a fragment of neurophysin II C-end glycopeptide) was isolated by A.A. Galoyan and coworkers in 1997, from the neurosecretory granules of bovine neurohypophysis, produced by the hypothalamic magnocellular nuclei. In intact rats galarmin-immunoreactive neurons and nerve fibers were widely distributed in the central nervous system. Single intramuscular injection of galarmin to the rats resulted in the increase of both galarmin-immunoreactivity and the number of galarmin-immunoreactive nerve cells, fibers and capillaries. In control experiments where the antisera against the fragment of immunophilin (a receptor of immunosuppressor macrolide FK-506) and the pancreatic neuropeptide Y were used as the primary antibodies, the significant increase of neuropeptide Y-immunoreactive nerve fibers and immunophilin-positive lymphocytes was revealed in galarmin-treated rats. Based on these results and the data on the motoneurons regeneration in the spinal cord hemisectioned rats given galarmin daily for 3 weeks, galarmin has been suggested to act as an immunomodulator, neurotransmitter and neuroregulator.

Vasopressin differentially modulates non-NMDA receptors in vasopressin and oxytocin neurons in the supraoptic nucleus.

Magnocellular neurons of the supraoptic nucleus release the neuropeptides oxytocin and vasopressin from their dendrites to regulate their synaptic inputs. This study aims to determine the cellular mechanism by which vasopressin modulates excitatory synaptic transmission. Presumably by electroporation through perforated patch, we were able to successfully introduce biocytin into cells in which we performed an electrophysiological study. This method enabled us to determine that roughly half of the recorded neurons were immunoreactive to oxytocin-associated neurophysin and showed two characteristic features: an inward rectification and a sustained outward rectification. The remaining half showed a linear voltage-current relationship and was immunoreactive to vasopressin-associated neurophysin. Using these electrophysiological characteristics and post hoc immunohistochemistry to identify vasopressin or oxytocin neurons, we found that vasopressin decreased evoked EPSCs in vasopressin neurons while increasing EPSCs in oxytocin neurons. In both types of neurons, EPSC decay constants were not affected, indicating that desensitization of non-NMDA receptors did not underlie the EPSC amplitude change. In vasopressin neurons, both vasopressin and a V1a receptor agonist, F-180, decreased AMPA-induced currents, an effect blocked by a V1a receptor antagonist SR49059. In oxytocin neurons, AMPA-induced currents were facilitated by vasopressin, whereas F-180 had no effect. An oxytocin receptor antagonist blocked the facilitatory effect of vasopressin. Thus, we conclude that vasopressin inhibits EPSCs in vasopressin neurons via postsynaptic V1a receptors, whereas it facilitates EPSCs in oxytocin neurons through oxytocin receptors.

Targeting the neurophysin-related cell surface antigen on small cell lung cancer cells using a monoclonal antibody against the glycopeptide region (MAG-1) of provasopressin.

The vasopressin (VP) gene is largely expressed in hypothalamic neurons, where the resultant pro-VP protein is enzymatically cleaved into its peptide hormone components, which include the neuropeptide VP, VP-associated neurophysin, and VP-associated glycopeptide (VAG). Small cell lung cancer (SCLC) tumors also express the VP gene, but the tumor pro-VP protein can remain intact and localize to the cell surface membrane. Previous studies have shown that polyclonal antibodies directed against different regions of the pro-VP protein bind specifically to the surface of cultured SCLC cells and recognize proteins of approximately 20 and approximately 40 kDa in cultured SCLC whole-cell lysate. Thus, these proteins have been designated neurophysin-related cell surface antigen (NRSA). A monoclonal antibody (mAb) designated MAG-1 was raised in this laboratory using a synthetic peptide representing the COOH-terminal sequence of VAG. The MAG-1 mAb recognizes NRSA in SCLC cell and tissue lysates by Western analysis, whereas immunofluorescent cytometric and microscopic analyses indicate that MAG-1 reacts specifically with NRSA on the surface of viable SCLC cells of both the classical and the variant subtype. Immunohistochemical analysis demonstrates that MAG-1 reacts with human SCLC tumor, but not with normal pulmonary epithelial cells in lung tissue. Additionally, a MAG-1 Fab fragment was generated that was also able to recognize NRSA. This is the first study to demonstrate that a mAb directed to the VAG region of the pro-VP protein has the potential for development into an in vivo diagnostic and therapeutic tool that targets plasma membrane-incorporated NRSA.

Membrane translocation and relationship with MHC class I of a human thymic neurophysin-like protein.

Thymic epithelial and nurse cells (TEC/TNC) synthesize an oxytocin (OT)-like peptide in association with a neurophysin (NP)-related protein in a way similar to in the hypothalamo-neurohypophysial (NHP) system. The central T-cell tolerance of the NHP neuroendocrine functions have been proposed to be mediated through these thymic NHP-related peptides due to their close homology with the NHP neurohormones OT and vasopressin (VP). In order to investigate their putative presentation by proteins of the major histocompatibility complex (MHC), human thymic membranes were purified and passed through an immunoaffinity column using mAb B9.12 directed to the monomorphic determinant of human MHC class I proteins. This methodology provided the following observations: (1) a NP-like protein is translocated in human thymic membranes and is retained by B9.12 on the column; (2) the MW of this NP-like material (50-55 kD) is quite different from the MW of hypothalamic NP proteins (10 kD), and (3) this thymic NP-like protein could be identified on Western blots with mAb B9.12. The precise extent of this relationship between the thymic NP-like protein and the Ig/MHC superfamily is actually investigated through the characterization of the genetic mechanisms responsible for the thymic expression of NHP-related peptides. Given the physiological importance of OT and of its binding to NP for transport along the axonal processes of the NHP tract, we postulate that, somewhat analogously, the thymic NP-/MHC class I-related protein could be involved in the presentation of the OT-like peptide to immature T-cells.

Histamine enhances the depolarizing afterpotential of immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus via H1-receptor activation.

Previous studies have demonstrated that histamine primarily excites unidentified neurons in the rat supraoptic nucleus. We investigated the neuromodulatory effects of histamine on immunohistochemically identified vasopressin neurons in the rat supraoptic nucleus using intracellular recording techniques from the hypothalamo-neurohypophysial explant. Exogenous application of histamine (0.1-100 microM) to vasopressinergic neurons produced a small membrane depolarization accompanied by an increase of up to 100% in the amplitude of the depolarizing afterpotential that follows current-evoked trains of action potentials. The enhancement of the depolarizing afterpotential by histamine did not depend upon the depolarization. Further, histamine enhanced the amplitude of the depolarizing afterpotential when blocking the afterhyperpolarizing potential with d-tubocurarine or apamin, and in the presence of tetrodotoxin and d-tubocurarine or apamin, indicating a postsynaptic action of histamine on the depolarizing afterpotential that is not simply a reflection of a decrease in the afterhyperpolarizing potential. These toxins also had no effect on the histamine-induced depolarization. The enhancement of the depolarizing afterpotential by histamine was mimicked by the histamine H1-receptor agonist 2-thiazolylethylamine and was reduced or blocked by the H1-receptor antagonist promethazine, but was not blocked or reduced in the presence of the histamine H2-receptor antagonist, cimetidine. In summary, these results show that the excitatory effect of histamine on immunohistochemically identified vasopressin neurons in the supraoptic nucleus is due in part to the H1-receptor-mediated enhancement of the depolarizing afterpotential independent of any change in the afterhyperpolarizing potential or membrane potential.

Vasopressin mRNA and neurophysin-related cell-surface antigen (NRSA) in small-cell carcinoma.

Production by small-cell carcinoma (SCCL) of neurophysins (HNPs) and neurophysin-related cell-surface antigen (NRSA) was examined for two cell lines, for mouse xenografts, and for a resected human tumor, using polyclonal and monoclonal antibodies to vasopressin-associated human neurophysin (VP-HNP) and polyclonal antibodies to vasopressin (VP). The nature of the mRNA responsible for giving rise to these neurophysin-related products was investigated by performing Northern analysis on preparations of poly A+RNA and cDNA probes complimentary to portions of the exon A, exon B, and exon C regions of the human VP gene. SDS-electrophoresis and Western analysis revealed two prominent proteins of 42,000 and 20,000 Da in acid extracts from all SCCL sources when the monoclonal anti-HNP or one of the two polyclonal anti-HNP preparations were used. These antibodies also disclosed the presence of a minor component of 10,000 Da. A second polyclonal anti-HNP preparation reacted with one prominent protein of 30,000 Da and, for one cell line and mouse xenografts, another protein of 32,000 Da. Both of two anti-VP preparations reacted with proteins of 42,000, 30,000, 25,000, and 20,000 Da in extracts from all SCCL source material. The immunoreactive proteins of 42,000, 30,000, and 20,000 Da were all components of a membrane fraction from SCCL cells and tissues. In Northern analysis, a single RNA of about 900 bases hybridized with exon A and exon B probes, but not with the cDNA probe complimentary to exon C of the VP gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Supraoptic nucleus afferents from the accessory olfactory bulb: evidence from anterograde and retrograde tract tracing in the rat.

Our earlier electrophysiological work provided evidence of a direct input to the supraoptic nucleus (SON) from the olfactory bulbs; however, these experiments could not determine if the input originated in the main and/or accessory portions of the olfactory bulb. Here, a connection between the accessory olfactory bulb (AOB) and the SON of the rat was examined using a combination of anatomic techniques. We employed neurophysin immunocytochemistry to delineate the morphological boundaries of the SON and the proximal arborizations of supraoptic dendrites. Accessory olfactory bulb efferents to the SON were studied by injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the AOB. The distribution of retrogradely labeled cells within the AOB was also determined after injection of either rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro-Gold (FG) into the SON. Neurophysin immunocytochemistry revealed that SON dendrites extended beyond the generally accepted boundaries of the nucleus, coursing ventrolaterally along the surface of the periamygdaloid cortex. Anterograde tract tracing with WGA-HRP labeled AOB efferents including a dense plexus of terminals and fibers around the ipsilateral SON along the path of the ventrally projecting dendrites. Injections of retrograde tracers into the SON resulted in rhodamine bead or FG labeling of mitral cells throughout the ipsilateral AOB. Taken together, these anatomic studies suggest a direct projection from the accessory olfactory bulb to the SON of the rat and thus a vomeronasal organ to SON pathway.

The recognition of hypothalamo-neurohypophysial functions by developing T cells.

Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.

Colocalization of immunoreactive oxytocin, vasopressin and interleukin-1 in human thymic epithelial neuroendocrine cells.

Monoclonal antibodies to oxytocin (OT) and vasopressin (VP) revealed some positively staining stromal cells in the subcapsular cortex and in the medulla of the human thymus. We further demonstrated that these cells are a subset of epithelial endocrine cells and also contain immunoreactive interleukin-1 together with the neuropeptides. In addition, the thymic cells stained by monoclonal antibodies directed to the cyclic part of oxytocin or vasopressin also contained some immunoreactive neurophysins. These data support the concept of intrathymic synthesis of neurohypophyseal-like peptides fitting the hypothalamic model. However, we observed that, contrary to the situation in the brain, OT- and VP-like peptides colocalized in the same thymic cells. Furthermore, one monoclonal antibody, specific for the tail part of oxytocin, did not label thymic cells. Therefore, thymic neuropeptide(s) could be related to, but distinct from, authentic OT and VP. These observations suggest some molecular differences between hypothalamic and thymic oxytocin biosynthetic pathways which need to be further investigated.

Expression of neurophysin-related precursor in cell membranes of a small-cell lung carcinoma.

A monoclonal antibody (mAb L6) to a small-cell lung carcinoma surface antigen recognizes a common epitope of vasopressin-neurophysin and oxytocin-neurophysin in hypothalamic nuclei. We now report on the identification of a neurophysin-like precursor in human lung carcinoma (LX-1) cell membrane. mAb L6 immunoaffinity chromatography of solubilized membranes resulted in a single band of approximately 45 kDa. Western blot analysis demonstrated immunoreactivity of this band with mAb L6, anti-vasopressin, and an antibody to the vasopressin precursor, pro-pressophysin. N-terminal sequencing of this band demonstrated a 21-amino acid homology with the N terminus of human pro-pressophysin, and substitution of a Cys33 residue in the tumor antigen with Arg33. Absence of immunoreactivity with the antibodies described above in cytosolic extracts and culture medium suggests nonsecretion of processed or intact pro-pressophysin-like peptide. Northern analysis of LX-1 mRNA with a 30-mer to the C terminus of rat pro-pressophysin resulted in a band of approximately 1000 base pairs, 250 base pairs larger than hypothalamic message. In situ hybridization of LX-1 tumor-bearing nude rat brain with the same probe demonstrated specific hybridization in rat hypothalamus and xenografted tumor. These findings suggest expression of a pro-pressophysin-like protein in this tumor cell line that is preferentially targeted to the cell membrane.

Imaging of small cell carcinoma using 131I-labelled antibodies to vasopressin associated human neurophysin (VP-HNP)

Preliminary studies are reported on the use of 131I-labelled antibodies against vasopressin associated human neurophysin to image tumors in patients with small cell carcinoma of the lung (SCCL). The rabbit polyclonal antibodies used in these studies were affinity purified on columns of neurophysin-Sepharose. Patients were pre-screened for the presence of neurophysin producing tumors by plasma RIA. Six patients who screened positive received approximately 1 mCi/70 kg body weight of radioiodinated antibody preparation, and scintigraphy was subsequently performed at 24 h, 48 h, and 72 h using a subtraction technique based on simultaneous imaging with radiopharmaceutical agents labelled with technetium-99m. Tumor was clearly demonstrated at 72 h in all five patients who had measurable lesions at the time of study, while no positive image was noted for one patient in complete clinical remission. Since approximately 70% of all SCCL patients have neurophysin-producing tumors, our findings suggest that neurophysin antibodies may be effectively used to scan tumors in a majority of patients with SCCL.

An antiserum to atrial natriuretic factor (ANF) cross-reacts with neurophysins in the hypothalamo-neurohypophysial system of rat brain.

The distribution of atrial natriuretic factor (ANF)-like reactivity was examined in rat brain and heart by immunohistochemistry. Immunostaining in heart was confined to atrial myocytes. In the hypothalamus, ANF-absorbable immunoreactivity was observed in magnocellular perikarya of the paraventricular and supraoptic nuclei, and in their projections to the neural lobe of the pituitary gland. No staining was seen in the preoptic or arcuate hypothalamic nuclei or in brain stem nuclei as previously reported by other investigators. The patterns of reactivity for ANF reported here is similar to that observed for neurophysins (NPs). Comparison of sequence data between rat ANF-28 and bovine NPs revealed three regions of 3 amino acid homology between these hypothalamic peptides. Preabsorption of the ANF antiserum with Affigel-coupled bovine NP I also resulted in complete elimination of all "ANF-immunoreactivity" in both atrium and hypothalamus. Cross-reactivity of the ANF antiserum with bovine NP I and II was further confirmed by Western blot analysis. Our findings suggest that ANF antisera can cross-react with NPs if they are directed against the shared antigenic epitopes; complete elimination of staining by preabsorption of the antibody with the immunogen, therefore, does not guarantee authenticity of localization. These observations may have relevance to an earlier study which reported on the existence of ANF-immunoreactivity in oxytocin neurons of the hypothalamus.

Vasopressin neuron is the target of monoclonal antibodies raised against vasopressin-neurophysin injected in vivo.

Monoclonal antibodies (MAbs) raised against the neurophysin (NP) specifically synthesized with vasopressin (VP, VP-NP) were injected into the paraventricular nucleus (PVN) of the rat hypothalamus. Their fate was studied by immunocytochemistry from 1 min to 3 h after the end of injection. It could be demonstrated that the VP-NP MAbs penetrated in vivo into some magnocellular neurons of the injected PVN and were transported ipsi- and contralaterally in individual neurons and in accessory magnocellular groups. When the time after injection was longer than 15 min, the VP-NP MAbs were also carried in the fibers of the median eminence. The prior treatment of rats with colchicine did not prevent the uptake of VP-NP MAb in the neurons but inhibited the transport towards the eminential fibers, the individual neurons and accessory groups. The detection of the PVN endogenous peptides (VP and oxytocin) on the same brain sections indicates that the neuronal uptake was specific. It only occurred in the neurons which synthesized VP and never appeared in the brain of rats suffering from a genetic defect of the central VP synthesis (Brattleboro rat). These data support the hypothesis of the location on the cell surface of the VP-NP precursor in magnocellular neurons which synthesize VP. This membrane signal identifies the neuron and allows the immunological recognition of the neurosecretory neurons in vivo.

Electron-microscopic immunocytochemistry of neuropeptide Y immunoreactive innervation of vasopressin neurons in the paraventricular nucleus of the rat hypothalamus.

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.

Developmental effect of dimethyl sulfoxide on hypothalamo-neurohypophysial neurons in vitro.

Primary dissociated cultures were established from diencephalic tissue of 14-day-old fetal rats. Neurons exhibiting immunocytochemical staining for neurophysin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio-immunometric quantitation of neuron-specific enolase. In contrast, in DMSO-treated cultures the number of neurophysin-immunoreactive neurons was more than doubled as compared to control cultures. [3H]Thymidine labeling and autoradiography in conjunction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time-course analysis of the action of DMSO revealed a 6-day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin-immunoreactive cells. These findings suggest that DMSO, which has previously been reported to have a differentiation-inducing effect on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.