Displayed Tyrosine Hydroxylase Immunoreactivity Research Articles

Expression and functions of β1- and β2-adrenergic receptors on the bulbospinal neurons in the rostral ventrolateral medulla.

The expression and effects of β-adrenergic receptors (β-ARs) on the neurons of the bulbospinal rostral ventrolateral medulla (RVLM) have been limitedly examined to date. The objective of this study was to examine the expression of β1- and β2-ARs on the bulbospinal RVLM neurons electrophysiologically and histologically. To directly investigate whether RVLM neurons display sensitivity to metoprolol (a β1-AR antagonist), dobutamine (a β1-AR agonist), butoxamine (a β2-AR antagonist), and salbutamol (a β2-AR agonist), we examined changes in the membrane potentials of the bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion of these drugs. During metoprolol superfusion, 16 of the 20 RVLM neurons were hyperpolarized, and 5 of the 6 RVLM neurons were depolarized during dobutamine superfusion. During butoxamine superfusion, 11 of the 16 RVLM neurons were depolarized, and all of the 8 RVLM neurons were hyperpolarized during salbutamol superfusion. These results suggest the expression of β1- and β2-ARs on the RVLM neurons. To determine the presence of β1- and β2-ARs histologically, immunofluorescence examination was performed. Five metoprolol-hyperpolarized neurons were examined for β1-AR and tyrosine hydroxylase (TH) immunoreactivity. All of the neurons displayed β1-AR immunoreactivity, whereas three of the neurons displayed TH immunoreactivity. All of the five RVLM neurons that became depolarized during metoprolol superfusion and hyperpolarized during butoxamine superfusion displayed β1- and β2-AR immunoreactivity. Our findings suggest that β1-AR antagonists or β2-AR agonists may decrease blood pressure through decreasing the activity of the bulbospinal RVLM neurons.

Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1β

It is well established that reciprocal modulation exists between the central nervous system and immune system. Interleukin (IL)-1beta, a proinflammatory cytokine secreted at early stage of immune challenge, has been recognized as one of the informational molecules in immune-to-brain communication. However, how this large molecule is transmitted to the brain is still unknown. In recent years it has been reported that the cranial nerves, especially the vagus, may play a pivotal role in this regard. It is proposed that IL-1beta may bind to its corresponding receptors located in the glomus cells of the vagal paraganglia and then elicit action potentials in the nerve. The existence of IL-1 receptor type I (IL-1RI) in the vagal paraganglia has been shown. The carotid body, which is the largest peripheral chemoreceptive organ, is also a paraganglion. We hypothesize that the carotid body might play a role similar to the vagal paraganglia because they are architectonically similar. Recently we verified the presence of IL-1RI in the rat carotid body and observed increase firing in the carotid sinus nerve following IL-1beta stimulation. The aim of this study was to observe the changes in expression of IL-1RI and tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, in the glomus cells of the rat carotid body following intraperitoneal injection of IL-1beta. The radioimmunoassay result showed that the blood IL-1beta level was increased after the intraperitoneal injection of rmIL-1beta (750 ng/kg) from 0.48+/-0.08 to 0.78+/-0.07 ng/ml (P<0.05). Immunofluorescence and Western blot analysis showed that the expression of IL-1RI and TH in the rat carotid body was increased significantly following peritoneal IL-1beta stimulation. In addition, double immunofluorescence labeling for TH and PGP9.5, a marker for glomus cells, or TH immunofluoresence with hematoxylin-eosin (HE) counterstaining revealed that a considerable number of glomus cells did not display TH immunoreactivity. These data provide morphological evidence for the response of the carotid body to proinflammatory cytokine stimulation. The results also indicate that not all of the glomus cells express detectable TH levels either in normal or in some abnormal conditions.

Dopaminergic projections from the VTA substantially contribute to the mesohabenular pathway in the rat

Recent evidence suggests that the lateral habenular complex (LHb) is a source of negative reward signals in midbrain dopaminergic neurons. LHb activity, in turn, is modulated by locally released dopamine, which is largely derived from the ventral tegmental area (VTA) via the mesohabenular pathway. Unfortunately, the presumed importance of this modulation has not been appreciated so far, as its intensity had been largely underestimated in previous reports. Consequently, the present study used contemporary techniques to reexamine the origin of dopaminergic fibers to the LHb. For this purpose, the retrograde tract-tracer gold-coupled wheatgerm agglutinin was injected into the LHb of fourteen rats. Four of these animals providing the most representative information were selected for detailed analysis. In total, 343 retrogradely labeled neurons were detected in the VTAs of these animals. By far most of them were found in the anterior VTA, accumulating in its ventral paramedian fields. About 47% (162) of retrogradely labeled cells displayed tyrosine hydroxylase immunoreactivity, suggesting that almost half of the mesohabenular neurons are dopaminergic. In addition, our data suggest that also incerto-hypothalamic and periventricular neurons contribute dopaminergic terminals to the LHb. The majority of LHb neurons, however, does not project to the origin of the mesohabenular pathway in the anterior VTA. Consequently, there might be no closed VTA-LHb-VTA loop. Instead, our data are in line with the idea that the anterior VTA via its projection to the medial part of the LHb may modulate the information flow from the limbic forebrain to monoaminergic midbrain nuclei.

Cyto- and Chemoarchitecture of Basal Forebrain Cholinergic Neurons in the Common Marmoset (Callithrix Jacchus)

The cyto- and chemoarchitecture of basal forebrain cholinergic neurons (BFCN) was investigated in the lower primate, the common marmoset (Callithrix jacchus). A large population of magnocellular, hyperchromic, and choline acetyltransferase (ChAT)-positive neurons was detected in the marmoset basal forebrain. The distribution of these neurons was similar to those in higher primates. Thus, ChAT-positive neurons were observed in the medial septum (Ch2), the vertical (Ch2) and horizontal (Ch3) limbs of the diagonal band of Broca, and the nucleus basalis of Meynert (Ch4). The Ch4 complex was relatively well differentiated and displayed distinct sectors. We detected anterior (Ch4a, with a medial and a lateral subdivision), intermediate (Ch4i, with a dorsal and a ventral subdivision), and posterior (Ch4p) sectors in the marmoset Ch4. The Ch4i was relatively small while the Ch4p was large. Similar to the rodent, the marmoset Ch1 extended quite a distance posteriorly, and the Ch4p displayed a major interstitial component distributed within the globus pallidus, its medullary laminae, and the internal capsule. Virtually all of the marmoset BFCN displayed acetylcholinesterase activity, and low affinity (p75NTR) and high affinity (Trk) neurotrophin receptor immunoreactivity. A majority contained immunoreactivity for calbindin-D28K and calretinin. Many of the Ch4 neurons also displayed tyrosine hydroxylase immunoreactivity. The BFCN lacked galanin immunoreactivity, but were innervated by galanin-positive fibers. None of the marmoset BFCN were NADPH-d-positive. Thus, the BFCN display major anatomical and biochemical differences in the marmoset when compared with higher primates. The marmoset BFCN also display many characteristics common to other primates. This fact, combined with the relatively short life span of the marmoset, indicates that this species may be ideal for studies of age-related changes in the BFCN.

Distribution of intrinsic cardiac neurons in whole-mount guinea pig atria identified by multiple neurochemical coding

Functional data indicate that neurons in distinct regions of the heart exert preferential regional cardiac control. To date the regional distribution of specific types of neurons within the intrinsic cardiac nervous system remains unknown, as does their associations with distinct neurotransmitter and/or neuromodulatory profiles. This study was designed to ascertain: (1) the distribution of different classes of neurons within the intrinsic cardiac nervous system as determined by microscopic analysis; (2) the neurochemical profiles of neurons in differing atrial loci; (3) which neurochemicals are co-localized within specific populations of intrinsic cardiac neurons; and (4) the distribution of specific sub-populations of neurons expressing specific immunoreactivities. Taking advantage of confocal laser scanning microscopy and distinct immunoreactive fluorescent markers in various double-label combinations, several sub-populations of intrinsic cardiac neurons were identified. Of all identified neurons, 85-90% were located in ganglia (ganglionic neurons), the rest being isolated (individual neurons). The two general neuronal markers protein gene product 9.5 (PGP 9.5) and microtubule-associated protein (MAP-2) were associated with neurons clustered primarily in the interatrial septum and around the origins of the two vena cavae. Ganglia (group 1) contained three sub-populations of neurons: approx. 80% of ganglionic neurons were large (15-40 microm diameters; group 1a) and approx. 20% had smaller diameters (less than 15 microm; group 1b). All of these neurons were PGP-immunoreactive, exhibiting choline acetyltransferase (ChAT) immunoreactivity (IR), tyrosine hydroxylase (TH) IR, neuropeptide Y (NPY) IR, vasoactive peptide (VIP) IR and substance P (SP) IR. The remaining 5% of ganglionic neurons were small (group 1c; less than 20 microm). These displayed TH immunoreactivity but not MAP, PGP, CHAT, NPY or SP immunoreactivity. Ten to fifteen percent of all neurons loosely distributed outside of ganglia were small (10-25 microm) and located primarily around the origin of the superior vena cava. They displayed immunoreactivity to TH, ChAT, VIP, NPY and SP, but not to MAP-2 or PGP 9.5. These data provide anatomical and immunohistochemical evidence for specific localization of differing populations of intrinsic cardiac neurons with respect to their size, ganglionic distributions and capacity to express multiple neurotransmitters. Although the functional importance of such a regional distribution of differing populations of intrinsic cardiac neurons remains unknown, these anatomical data support the thesis that unique clustering of specific populations of neurons within this nervous system represents the anatomical substrate for complex local cardiac regulatory phenomena occurring at the level of the target organ.

Localization of GABA transaminase immunoreactivity in the rat substantia nigra pars reticulata

Precise cellular localization of gamma-aminobutyric acid transaminase (GABA T), a degrading enzyme for the neurotransmitter GABA, was determined in the rat substantia nigra (SN) by immunocytochemical experiments using a recently developed monoclonal antibody. In order to characterize the GABA T-immunoreactive neurons, double immunocytochemistry was also performed using tyrosine hydroxylase (TH) as a neurochemical marker for dopaminergic neurons in the substantia nigra pars compacta (SNc). Immunoreactivity for GABA T was primarily localized in perikarya of the SN. There were only a few GABA T-immunoreactive neurons found to display TH immunoreactivity. Most of the GABA T-immunoreactive neurons were then identified as reticulata neurons. These results indicate that reticulata neurons are the major nigral neurons that express GABA T immunoreactivity and there may be functional compartmentalization of the GABA metabolism in the rat substantia nigra pars reticulata (SNr).

Brainstem dopaminergic, cholinergic and serotoninergic afferents to the pallidum in the squirrel monkey

The retrograde tracer cholera toxin B subunit (CTb) was used in combination with immunohistochemistry for tyrosine hydroxylase (TH), calbindin D-28k (CaBP), choline acetyltransferase (ChAT) and 5-hydroxytryptamine (5-HT) to determine the distribution and relative proportion of brainstem chemospecific neurons that project to the pallidum in the squirrel monkey ( Saimiri sciureus). Large injections of CTb involving both pallidal segments produce numerous retrogradely labeled neurons in the substantia nigra (SN), the pedunculopontine tegmental nucleus (PPN) and the dorsal raphe nucleus (DR). Labeled neurons are distributed uniformly in SN with a slight numerical increase at the junction between the pars compacta (SNc) and the ventral tegmental area (VTA). Retrogradely labeled neurons abound also in PPN, principally in its pars dissipata, whereas other CTb-labeled cells are scattered throughout the rostrocaudal extent of DR. After CTb injection involving specifically the internal pallidal segment (GPi), the same pattern of cell distribution is found in SN, PPN and DR, except that the number of retrogradely labeled cells is lower than after large pallidal complex injections. Approximately 70% of all CTb-labeled neurons in SNc-VTA complex display TH immunoreactivity, whereas 20% are immunoreactive for CaBP. About 39% of all retrogradely labeled neurons in PPN are immunoreactive for ChAT, whereas approximately 38% of the labeled neurons in DR display 5-HT immunoreactivity. Following CTb injection in the external pallidal segment (GPe), the number of labeled cells is much smaller than after GPi injection. The majority of CTb-labeled cells in SNc-VTA complex are located in the lateral half of SNc and approximately 93% of these neurons display TH immunoreactivity compared to 10% that are immunoreactive for CaBP; very few CTb-labeled cells occur in PPN. Retrogradely labeled cells in DR are located more laterally than those that projects to the GPi and about 25% of them are immunoreactive for 5-HT. These results suggest that, in addition to their action at striatal and/or nigral levels, the brainstem dopaminergic, cholinergic and serotoninergic neurons influence the output of the primate basal ganglia by acting directly upon GPi neurons.

Innervation of the amygdaloid complex by catecholaminergic cell groups of the ventrolateral medulla.

The projections to the amygdaloid complex (AMG), originating in the catecholaminergic cell groups of the ventrolateral medulla (VLM), were studied in the rat by using either the retrograde tracer fluoro-gold (FG) or the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with tyrosine hydroxylase (TH) and/or phenylethanolamine-N-methyltransferase (PNMT) immunohistochemistry. In the first series of experiments, injections of FG were made into regions of the central nucleus of the amygdala (ACe) where dense TH and PNMT immunoreactivity was previously observed, and then sections of the brainstem were processed for TH and PNMT immunoreactivity. FG retrogradely labelled neuronal cell bodies were observed throughout the rostrocaudal extent of VLM, bilaterally, with a contralateral predominance. Approximately 44% of the FG labelled cell bodies in VLM were also immunoreactive to the catecholamine biosynthetic enzymes TH and/or PNMT. Most of these catecholaminergic neurons were part of the A1 noradrenergic cell group in the caudal VLM and to a lesser extent part of the C1 adrenergic cell group in the rostral VLM. In the second series of experiments, PHA-L was iontophoresed into VLM at different rostrocaudal levels where in the previous series of experiments FG retrogradely labelled cell bodies were observed. Transverse sections of the forebrain and brainstem were then processed for the demonstration of PHA-L and either TH or PNMT immunoreactivity in cell bodies, axons, and presumptive axon terminals. PHA-L injection sites within either the caudal or rostral VLM resulted in labelled axons and terminal bouton-like swellings primarily in the contralateral AMG and to a lesser extent in the ipsilateral AMG. The ACe was observed to receive the greatest innervation from either VLM site. Additionally, PHA-L labelled fibers and presumptive terminal boutons were observed within the intercalated, medial, basomedial, and basolateral nuclei of the AMG. Most of the PHA-L labelled fibers and presumptive terminal boutons in the AMG after a caudal VLM (A1 region) injection also displayed TH immunoreactivity, whereas after a PHA-L injection into the rostral VLM (C1 region) all of the labelled axons and axon terminals in the AMG also were immunoreactive to PNMT. These data demonstrate that catecholaminergic neurons in A1 and C1 regions of VLM innervate the AMG and suggest that these VLM neurons may be involved in relaying afferent information directly to the AMG which influences the activity of AMG neurons controlling autonomic, endocrine, and behavioural functions.

A novel population of tyrosine hydroxylase immunoreactive neurones in the basal forebrain of the common marmoset ( Callithrix jacchus)

We have observed in the basal forebrain of the common marmoset a group of neurones which display tyrosine hydroxylase immunoreactivity (THir) with three different polyclonal antibodies and one monoclonal antibody, and which express TH mRNA as shown by in situ hybridization histochemistry. The population of cells is composed of large multipolar neurones and is located predominantly in the substantia innominata and at the ventral, medial and lateral margins of the external segment of the globus pallidus. The cell morphology and the distribution of THir cells corresponds closely to the caudal portion of the nucleus basalis of Meynert. Adjacent sections demonstrate both THir and choline acetyltransferase immunoreactivity in the cells in this group, as well as strong acetylcholinesterase activity but not dopamine immunoreactivity. These observations indicate that many cholinergic neurones in the posterior nucleus basalis of Meynert of the marmoset contain tyrosine hydroxylase, and suggest that both acetylcholine and catecholamine may be synthesised as co-localised neurotransmitters within the same magnocellular neurones. We observe no THir cells in similar areas of the basal forebrain of either rhesus or talapoin monkeys.

Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: direct evidence for noradrenergic and cholinergic properties in the same neurons.

Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non-overlapping populations, and (2) to what extent the levels of synthesis are influenced by non-neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non-neuronal cells purified by fluorescence-activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non-neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH-positive neurons are recognized by an anti-ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non-neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated.

Non-dopaminergic projection from the subparafascicular area to the temporal cortex in the rat

Following injection of fluorescent tracer into the rat temporal cortex, retrogradely labeled cells were found in the subparafascicular area. In the thalamus, numerous cell labeling was seen in the medial geniculate complex. Labeled cells in the subparafascicular area were small (10–20 μm), and did not display tyrosine hydroxylase immunoreactivity. From these findings, the subparafascicular area has, at least, two distinct projection systems towards the cerebral cortex; the previously described large dopaminergic cells innervating the frontal cortex and small non-dopaminergic cells innervating the temporal cortex.

Generation of developmental patterns in the neuroepithelium of the developing mammalian eye: The pigment epithelium of the eye

Culture experiments with eye anlages of mouse embryos were performed to study developmental traits of the neuroepithelial cells of the prospective pigment epithelium in the eye anlage of pigmented mice. Between the neural plate stage on embryonic day 8 (ED 8, developmental stage 12) and the neural tube stage on embryonic day 9 1 2 (stage 15), the cultured neuroepithelium of the eye generated neurons and glia, identified by morphological and immunocytochemical evidence, but no pigmented cells. In contrast, eye anlages did produce pigment epithelium when cultured in their natural position in a head tissue fragment. A minority of developing neurons displayed tyrosine hydroxylase immunoreactivity, whereas GABAergic, serotoninergic and substance P-ergic neurons, which are common in the mature neuroretina, were not observed. When neuroepithelial cells from embryonic eyes older than stage 15 (ED 9 1 2 ) were cultured, they differentiated into pigment cells but not into nerve cells or glia. This developmental sequence indicates that the pigment cells derive from the neural lineage. Pigment cell fate dominates over the neural fate beginning at about stage 15 (ED 9 1 2–10 ). That is at least 2 days before the pigment cell phenotype becomes apparent in vivo (ED 11 1 2–12 ).

Evidence for a distinct nigropallidal dopaminergic projection in the squirrel monkey

Injections of the retrograde fluorescent tracer fast blue in the striatum (STR) and nuclear yellow in the internal segment of the globus pallidus (GPi) in the squirrel monkey ( Saimiri sciureus) revealed a nigropallidal projection whose cellular origin was largely distinct from that of the nigrostriatal pathway. Neurons containing the tracer injected in GPi were scattered throughout the substantia nigra-ventral tegmental area complex where they formed approximately 20–25% of the total number of retrogradely labeled cells. Only about 5–10% of all positive neurons were double-labeled after STR-GPi injections. In experiments combining the use of the fluorescent tracer propidium iodide with immunofluorescence, the majority of neurons projecting to GPi displayed tyrosine hydroxylase immunoreactivity. Hence, in addition to their important role at striatal level, midbrain dopaminergic neurons may influence directly the output neurons of the basal ganglia at pallidal level in primates.

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches.

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.