Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro

Summary In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently of the action of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.     

Localization of tyrosine hydroxylase immunoreactive neurons in the forebrain of the guppy Poecilia reticulata

The current study reports for the first time the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the forebrain of the guppy Poecilia reticulata . Numerous small TH-ir neurons were observed in the olfactory bulbs, located mainly in the periphery of the bulbs. The TH-ir telencephalic neurons are localized in the ventral telencephalic area where they are grouped in three distinct nuclei (Vv,Vd and Vp) composed of a small number of cells forming a continuous strip. The largest number of forebrain TH-ir neurons was observed in the diencephalon where both small and larger neurons are present. Diencephalic TH-ir neurons are subdivided in large nuclei located in the preoptic region (nSC, nPOp and nPOm), the thalamus (nDM), the pretectal region (nPPv and nAP), the hypothalamus (nPP and nRP) and the posterior tuberculum (nPT). Many diencephalic nuclei are distributed in periventricular regions and no TH-ir cells were observed in the paraventricular organ. A comparative analysis indicates that the present observations are consistent with the general pattern of TH-ir neurons distribution reported for the forebrain of other teleosts, but with some interspecies variability present, mainly in the diencephalon. This paper also provides valuable neuroanatomical information for P. reticulata , a teleost frequently used in toxicological tests, for future studies investigating the effects of environmental pollutants on the catecholaminergic system.     

Development of neuropeptide Y and tyrosine hydroxylase immunoreactive innervation in postnatal rat heart

Neuropeptide Y (NPY), immunoreactive (IR), and tyrosine hydroxylase (TH)-IR nerve fibers were scarce at birth in rat heart, but increased rapidly during the first 2 postnatal weeks, reaching approximately adult levels by the third week. The sequence of development was: interatrial septum and atrial wall, free ventricular wall starting from the epicardium, and finally the atrial appendages and interventricular septum. In ventricles and atrial appendages both fiber types developed similarly. In interatrial septum and atrial walls more NPY-IR than TH-IR fibers were evident, and NPY-IR, but not TH-IR, neurons were detected in intrinsic ganglia. Doublelabel immunohistochemistry provided further evidence that NPY is located in ventricular and atrial noradrenergic nerves, but is also located in nonnoradrenergic nerves in atria.     

Cytoplasmic microtubules of normal and tumor cells of the leopard frog

Abstract. The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28° C; similar results were obtained at 20° C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7° C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28° C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 7° C. However, while normal cells could effect a recovery at 7° C after the removal of nocodazole, PNKT4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28° C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7° C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.     

Species differences in the expression of multiple tyrosine hydroxylase protein isoforms

In subprimates, a single form of tyrosine hydroxylase (TH) is expressed, whereas two TH protein isoforms have been identified in monkeys and four isoforms have been demonstrated in humans. In order to establish the evolutionary pattern/emergence of these multiple TH isoforms, adrenal medullae from different mammalian species were analyzed by blot immunolabeling using pan-specific TH antibodies and antibodies specific to each of the four human TH isoforms. The expression of multiple TH isoforms was primate specific and restricted to anthropoids: only a single TH isoform was detected in adrenal medullae from several subprimate and prosimian species (six species from four families), while two TH isoforms were found in all of the anthropoid species studied. The presence of four TH isoforms could only be demonstrated in human specimens. Contrary to previous suggestions, only one TH protein isoform was found in rats and only four TH protein isoforms were found in humans.     

Dopamine beta-hydroxylase like immunoreactive cells in the frog taste disc

We immunohistochemically examined the existence of dopamine beta-hydroxylase (DBH), a noradrenalin (NA)-synthesizing enzyme from dopamine, in the taste disc of frog, Rana catesbeiana. DBH-like immunoreactive cells were located in the intermediate layer in the taste disc; the cells showed an apical process reaching the surface of the disc and one or several basal processes. Cells with a thick apical process and those with a thin apical process were both immunoreactive: these cells corresponded to type II and III receptor cells of the frog taste disc. Immunoreactive granules were observed in the cytoplasm of those cells. In the frog taste disc, only type III cells are reported to have afferent synapses with the nerve via basal processes but those basal processes have not been reported in type II cells. In the present study, we found that type II-like cells possessed a long basal process extending toward the basal lamina. Mucous (type Ia) cells, wing (type Ib) cells, and glia-like sustentacular (type Ic) cells were all immunohistochemically unreactive. The present observations support the argument that NA (or adrenalin) may work as a chemical transmitter in the frog taste organ.     

Species differences in the regulation of tyrosine hydroxylase in Cnemidophorus whiptail lizards

Evolution of behavioral phenotype involves changes in the underlying neural substrates. Cnemidophorus whiptail lizards enable the study of behavioral and neural evolution because ancestral species involved in producing unisexual, hybrid species still exist. Catecholaminergic systems modulate the expression of social behaviors in a number of vertebrates, including whiptails, and therefore we investigated how changes in catecholamine production correlated with evolutionary changes in behavioral phenotype by measuring the size and number of catecholamine producing (tyrosine hydroxylase-immunoreactive, or TH-ir) cells across the reproductive cycle in females from two related whiptail species. Cnemidophorusuniparens is a triploid, parthenogenetic species that arose from hybridization events involving the diploid, sexual species C. inornatus. Prior to ovulation, females from both species display femalelike receptive behaviors. However, after ovulation, only parthenogenetic individuals display malelike mounting behavior. In all nuclei measured, we found larger TH-ir cells in the parthenogen, a difference consistent with species differences in ploidy. In contrast, species differences in the number of TH-ir cells were nucleus specific. In the preoptic area and anterior hypothalamus, parthenogens had fewer TH-ir cells than females of the sexual species. Reproductive state only affected TH-ir cell number in the substantia nigra pars compacta (SNpc), and C. uniparens individuals had more TH-ir cells after ovulation than when previtellogenic. Thus, species differences over the reproductive cycle in the SNpc are correlated with species differences in behavior, and it appears that the process of speciation may have produced a novel neural and behavioral phenotype in the parthenogen.     

Tympanic and extratympanic sound transmission in the leopard frog

Summary The inner ear of the leopard frog,Rana pipiens, receives sound via two separate pathways: the tympanic-columellar pathway and an extratympanic route. The relative efficiency of the two pathways was investigated. Laser interferometry measurements of tympanic vibration induced by free-field acoustic stimulation reveal a broadly tuned response with maximal vibration at 800 and 1500 Hz. Vibrational amplitude falls off rapidly above and below these frequencies so that above 2 kHz and below 300 Hz tympanic vibration is severely reduced. Electrophysiological measurements of the thresholds of single eighth cranial nerve fibers from both the amphibian and basilar papillae in response to pure tones were made in such a way that the relative efficiency of tympanic and extratympanic transmission could be assessed for each fiber. Thresholds for the two routes are very similar up to 1.0 kHz, above which tympanic transmission eventually becomes more efficient by 15–20 dB. By varying the relative phase of the two modes of stimulation, a reduction of the eighth nerve response can be achieved. When considered together, the measurements of tympanic vibration and the measurements of tympanic and extratympanic transmission thresholds suggest that under normal conditions in this species (1) below 300 Hz extratympanic sound transmission is the main source of inner ear stimulation; (2) for most of the basilar papilla frequency range (i.e., above 1.2 kHz) tympanic transmission is more important; and (3) both routes contribute to the stimulation of amphibian papilla fibers tuned between those points. Thus acoustic excitation of the an uran's inner ear depends on a complex interac tion between tympanic and extratympanic sound transmission.Abbreviations dB SPL decibels sound pressure level re: 20 N/ m² - AP amphibian papilla - BP basilar papilla - BEF best excitatory frequency     

Differential distribution of a second type of tyrosine hydroxylase immunoreactive amacrine cell in the chick retina

A second population of tyrosine hydroxylase-immunoreactive amacrine cells was demonstrated in embryonic and adult chicken retinas by immunohistochemistry techniques in whole flat-mount preparations. The populations were differentiated on a basis of different immunostaining intensities, levels of stratification in the inner plexiform layer, and topographical distributions. Cells of one type were similar to the previously described dopaminergic amacrine cells, denoted here as tyrosine hydroxylase type 1 cells. Immunoreactive neurons of the second type observed in the present work had relatively smaller somata size, and weaker immunostaining than type 1 cells, and were located preferentially in the ventral retina. These tyrosine hydroxylase type 2 cells could be visualized from embryonic day 14 to 21 days after hatching animals. The distribution of the second population was coincident with that of the targets of centrifugal fibres and with cells involved in long proprioretinal connections. We propose that the tyrosine hydroxylase type 2 amacrine cells found in the ventral retina could mediate an important pathway to the upper half of the visual field so as to aid in the detection of predators.     

Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Temperature effects

The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28 degrees C; similar results were obtained at 20 degrees C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7 degrees C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28 degrees C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 70 degrees C. However, while normal cells could effect a recovery at 7 degrees C after the removal of nocodazole, PONKT-4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28 degrees C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7 degrees C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.     

Partial coexistence of growth hormone-releasing hormone and tyrosine hydroxylase in paraventricular neurons in rats

Immunocytochemistry revealed growth hormone-releasing hormone (GRF)-containing cells (4-10/50 microns thick coronal sections) in the ventral portion of the medial parvicellular subdivision of the paraventricular nucleus (PVN) in the rat. In the same region we also detected about the same number of neurons containing the mRNA that encodes GRF using in situ hybridization histochemistry. Fluorescence double labelling immunohistochemistry showed the presence of GRF and tyrosine hydroxylase (TH) in the same PVN neurons. The overlap between the two populations of cells is only partial: less than half of TH-positive cells contain GRF and vice versa.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

Electron microscopic analysis of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80-120 nm dense core granules and 30-50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons contained 70-100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN. Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN. In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70-120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons. These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.     

Adaptive changes of H+ secreting cells in the epidermis of the leopard frog Rana pipiens

1. Mitochondria-rich (MR) cells in the integument of the southern leopard frog, Rana pipiens, berlandieri, were stained with AgNO3 under a variety of environmental and metabolic treatment conditions known to increase H+ excretion rates across the skin. In this tissue AgNO3 proved to be a good stain for discriminating the MR cell populations from the granular cells. 2. High salinity adapted southern frogs showed no change in the MR cell population. The inability of the MR cell number to significantly increase suggested that the increased H+ excretion rates previously seen in these animals were not due to increased MR cell proliferation. 3. The MR cell population was found to increase in the NaNO3 adapted frogs, demonstrating the contribution of altered extracellular Cl- concentrations on the regulation of MR cell density. 4. Animals that were placed in chronic metabolic acidosis or pre-treated with ibuprofen demonstrated an increased MR cell population. The current observations are consistent with previous findings that these treatment regimes increase H+ excretion, suggesting that one of the cellular adaptive mechanisms responsible for increasing H+ excretion involves increasing the MR cell density. 5. The results further suggest that prostaglandins may play a role in regulating H+ excretion in MR cells, and that either changes in intracellular pH or prostaglandin formation regulates cell proliferation.