Effects of a novel differentiation factor on the development of catecholamine traits in noncatecholamine neurons from various regions of the rat brain: studies in tissue culture.

The muscle-derived differentiation factor called MDF initiated expression of the catecholamine (CA) enzyme tyrosine hydroxylase (TH) in non-CA neurons isolated from a variety of regions in the rat brain. Specifically, subpopulations of neurons from the striatum, collicular plate, and cerebellum were TH-immunoreactive after an overnight exposure to MDF in culture. The number of immunopositive cells was greatest in the striatum, where more than half of all plated neurons expressed the enzyme. In contrast, MDF had no effect on the central neurons of the hippocampus or on peripheral sensory neurons. In 3H-thymidine studies, only brain neurons that had already withdrawn from mitosis expressed TH. These cells remained open to the epigenetic influence of MDF only during a brief and defined critical period that appears to be timed intrinsically. Without daily replenishment of MDF, expression of the enzyme disappeared after several days in culture, suggesting that MDF was rapidly depleted or degraded in vitro. However, in the continued presence of MDF, TH expression was maintained indefinitely, thus producing a permanent alteration in phenotype. Moreover, a single exposure to MDF during the critical period was sufficient to render neurons permanently receptive to the molecule so that TH expression could be reinitiated many days later. It is postulated that a memory of this biochemical interaction was established in these neurons, making transmitter phenotypic plasticity possible at later stages.     

A muscle-derived factor(s) induces expression of a catecholamine phenotype in neurons of cultured rat cerebral cortex

We sought to determine the source of the signal(s) that promotes expression of the catecholamine (CA) enzyme tyrosine hydroxylase (TH) in cultured neurons of embryonic rat cerebral cortex, a tissue which is not thought to contain CA cells in vivo. Cortical neurons were cultured with their non-neuronal constituents and 48 hr later immunostained for TH. Fibroblasts or glia had no effects, however, blood vessels increased the numbers of TH neurons nearly 4-fold. Coculture with either perinatal aorta, skeletal or cardiac muscle, clonal muscle cell lines 1440 (smooth) and L6 (skeletal), conditioned media from L6 cells, or a soluble extract of L6 cells increased the number of TH neurons up to 20-fold. The induction of TH by muscle extract was (1) dose dependent; (2) paralleled by a proportional increase in the steady-state levels of TH mRNA; (3) greatly reduced by the RNA synthesis inhibitor alpha-amanitin or the protein synthesis inhibitor cycloheximide; and (4) unassociated with change in the survival of neurons in culture. The response was not replicated by treatment with other established neurotrophic substances, including NGF, EGF, FGF, PDGF, neuroleukin, insulin, pyruvate, KCI, adenosine, or inosine. We conclude that muscle contains a potentially novel substance, muscle-derived differentiation factor (MDF) that promotes differentiation but not survival of neurons of cerebral cortex by de novo synthesis of TH mRNA and TH protein. Thus, neurons of the CNS, as in periphery, may undergo phenotypic interconversion in response to biologically derived molecules in their environment.     

Effects of perinatal exposure to Δ9-tetrahydrocannabinol on the fetal and early postnatal development of tyrosine hydroxylase-containing neurons in rat brain

The exposure of pregnant rats to Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive constituent ofCannabis sativa, during the perinatal period affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brains of their offspring at peripubertal and adult ages. In the present work we explored whether these effects also appear during fetal and early neonatal periods, when TH expression plays an important role in neural development. To this end, the mRNA amounts for TH and the amounts and activity of this enzyme, in addition to catecholamine (CA) contents, were analyzed in the brain of fetuses at different gestational days (GD) and of newborns at two postnatal ages, which had been daily exposed to Δ⁹-THC or vehicle from d 5 of gestation. Results were as follows. The exposure to Δ⁹-THC markedly affected the expression of the TH gene in the brain of fetuses at GD 14. Thus, the amounts of its mRNA at this age were higher in Δ⁹-THC-exposed fetuses than in controls. This corresponded with a marked rise in the amounts of TH protein and in the activity of this enzyme at this age. Normalization was found in these parameters at GD16. However, a marked sexual dimorphism in the response of TH gene to cannabinoid exposure appeared from GD18 and was particularly evident at GD21, when TH-mRNA amounts increased in developing female brains, but decreased in developing male brains exposed to Δ⁹-THC, effects that were mostly prolonged to early postnatal ages. However, these changes did not correspond always with parallel changes in the amounts and activity of TH and in CA contents, as occurred in GD14, suggesting that Δ⁹-THC would not be affecting the basal capability to synthesize CAs in TH-containing neurons, but would affect the responsiveness of TH gene. We found only a marked increase in the production ofl-3,4-dihydroxyphenylacetic acid, the main intraneuronal dopamine metabolite, in female newborns exposed to Δ⁹-THC. Collectively, our results support the belief that the perinatal exposure to Δ⁹-THC affects the expression of the TH gene and, sometimes, the activity of this enzyme in brain catecholaminergic neurons in certain critical periods of fetal and early neonatal brain development. These results support the notion that cannabinoids are able to affect the gene expression of specific key proteins for catecholaminergic development, and that these alterations might be the origin of important long-term neurobehavioral effects caused by perinatal cannabinoid exposure at peripubertal and adult ages. Part of this work has been previously presented in abstract form and published in the Proceedings of the International Cannabis Research Society (July 21–23, 1994, L’Esterel, Quebec, Canada).     

Expression and regulation of catecholaminergic traits in primary sensory neurons: relationship to target innervation in vivo

Catecholaminergic (CA) phenotypic characteristics have recently been detected in adult sensory neurons, demonstrating that CA expression in the periphery extends beyond the sympathoadrenal axis. Consequently, we may now determine whether common principles underlie CA phenotypic organization in functionally and embryologically diverse populations of peripheral neurons. To begin defining sensory transmitter regulation, the present study examined the relationship of CA expression to sensory target innervation in cranial nerve ganglion cells of the adult rat. Retrograde labeling combined with tyrosine hydroxylase (TH) immunocytochemistry indicated that 80-90% of CA sensory neurons in the glossopharyngeal petrosal ganglion project peripherally in the carotid sinus nerve (CSN). Most of these cells innervate a single target, the carotid body, revealing a striking correlation between CA expression and the pattern of sensory target innervation. Furthermore, CSN transection resulted in a transient marked decrease in TH catalytic activity and immunoreactivity within 1 week. Activities returned to normal by 3 weeks. Thus, axotomy reversibly decreased sensory TH, reproducing effects observed with central CA neurons (Ross et al., 1975), but differing in certain aspects from observations with sympathetic CA neurons (Cheah and Geffen, 1973; Kessler and Black, 1979). To determine whether disruption of axonal transport itself decreased TH in petrosal neurons, colchicine cuffs were placed around the intact CSN. Colchicine blockade reproduced the effects of axotomy, suggesting that deranged transport, and not axonal damage per se, altered TH. Finally, we studied the role of sensory projections to the CNS by examining petrosal TH after glossopharyngeal nerve rhizotomy. In contrast to sequelae of peripheral axotomy, rhizotomy did not alter TH, suggesting that projections to the periphery predominate in regulation of sensory TH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of retinoic acid on NB 69 human neuroblastoma cells and fetal rat mid brain neurons

Summary Retinoids are chemical compounds which play important roles in ontogenetic development and cranio-caudal differentiation in animals, but their effect on phenotypic expression of neurotransmitters are unknown. We studied the pharmacological and morphological effects of retinoic acid (RA) on two types of immature vertebrate neurons, the human derived neuroblastoma cells, NB69, and fetal rat mid brain neurons in culture. The pharmacological effects of RA on the cultures and their relation to catecholamine and acetylcholine neurotransmission were evaluated according the levels of catecholamines, tyrosine hydroxylase (TH) activity, TH immunostaining, and choline acetyltransferase (CAT) activity, respectively. RA reduces catecholamine levels and TH activity in NB69 cells and the number of dopamine neurons in cultures derived from rat fetal mid brain. The detrimental effect of RA on mid brain neurons is dose- dependent; limited to TH+ cells at low concentrations (100 to 500 nM) and toxic for all types of cells at high concentrations (1 to 2 M). RA increases CAT activity in NB 69 cells and produces phenotypic differentiation of these to a more mature neuronal phenotype with more prolonged neurite extensions. Therefore, RA may play a trophic positive role in the differentiation of immature cells to cholinergic neurons; this contrasts with the detrimental effects of RA on catecholamine neurons.     

Inducible Expression of Tryptophan Hydroxylase without Serotonin Synthesis in Hypothalamic Dopaminergic Neurons

In the present study we have further studied the previous findings that rat hypothalamic dopaminergic neuronal cell groups may express tryptophan hydroxylase (TpH), the serotonin synthesizing enzyme, without a detectable serotonin synthesis. Chemical and mechanical neuronal injuries, namely colchicine treatment and axonal transection, respectively, were performed, and distributions of neurons exhibiting immunoreactivity for TpH and/or tyrosine hydroxylase (TH), the dopamine synthesizing enzyme, were analyzed throughout the hypothalamic periventricular and arcuate nuclei. After colchicine treatment there was a statistically significant 87% (P = 0,01) increase in the number of TpH expressing neurons, while TH expression remained essentially similar. Axonal transection resulted also in a statistically significant 131% (P < 0,01) increase in the number of TpH expressing neurons, while TH expression was not significantly altered. All TpH expression coexisted with TH expression, and the induction of TpH expression by neuronal injuries occurred evenly throughout the rostrocaudal length of the territory studied. A possible serotonin synthesis by TpH was examined by giving drugs that increase brain serotonin synthesis, but no immunohistochemically detectable serotonin synthesis could be found in any of the TpH expressing neurons. Finally the possibility was studied that the relative shortage of the cofactor tetrahydrobiopterin would limit serotonin synthesis. However, an administration of tetrahydrobiopterin did not result in detectable serotonin synthesis in these neurons. Taken together these results suggest that dopaminergic neurons in the hypothalamic periventricular and arcuate nuclei are able to express TpH, this expression is induced after neuronal injury, and this induction occurs similarly throughout the territories studied. TpH expression occurs independently of TH expression, and the newly expressed TpH appears not to synthesize serotonin, regardless of pharmacological pretreatments. Thus, our findings (i) support the idea that neurons may possess inducible expression of nonfunctional transmitter-synthesizing enzymes, in this case TpH, and (ii) suggest that expression of an enzyme synthesizing a certain transmitter may not necessarily imply the corresponding transmitter phenotype.     

The role of GABA in the regulation of the dopamine/tyrosine hydroxylase-containing neurons of the rat retina

Dopamine (DA) and gamma-aminobutyric acid (GABA) are putative neurotransmitters in two separate populations of amacrine neurons in the mammalian retina. Pharmacological studies have been conducted to determine if GABA neurons regulate the neuronal activity of the neurons that secrete DA. Tyrosine hydroxylase (TH) activity, a biochemical indicator of changes in activity of DA/TH-containing neurons, was low in dark-adapted retinas and high in light-exposed retinas. Muscimol (a GABA receptor agonist) produced a dose-related, biphasic effect on the light-evoked activation of TH, when the drug was injected into the vitreous (intravitreal injection) of dark-adapted rats. At low doses, (35 and 60 pmol) muscimol enhanced the light-evoked activation of TH, but at higher doses (greater than or equal to 120 pmol) it inhibited the light-evoked increase in enzyme activity. Muscimol had no significant effect on the TH activity of dark-adapted retinas. GABA antagonists, bicuculline and picrotoxin, produced effects on TH activity that were dependent on both dose and light-exposure. At low doses (0.4-0.5 nmol), bicuculline and picrotoxin both inhibited the light-evoked activation of TH, but had no effect on TH activity of the dark-adapted retinas. At a higher dose (2.0 nmol), both antagonists increased TH activity in the dark-adapted retina and attenuated the further activation of the enzyme by light. Rat retinas were dissociated into suspensions of viable cells in order to investigate the direct effects of muscimol and picrotoxin on the DA/TH-containing cells. The process of dissociating dark-adapted retinas resulted in an apparent activation of TH. Incubation of the cells with muscimol resulted in a decrease of TH activity in a concentration-dependent manner. Picrotoxin antagonized the inhibitory effect of muscimol, but had no effect when incubated alone. The biphasic effects of GABA agonists and antagonists in vivo suggest that a certain subpopulation of GABA neurons are involved in the activation of the DA/TH-containing neurons by photic stimulation, while another subpopulation of GABA neurons produce a tonic inhibition of the DA/TH-containing neurons in darkness. The experiments with retinal cell suspensions indicate that the tonic inhibition is probably mediated by synapses of GABA neurons directly onto the DA/TH-containing cells.     

A new double labeling technique using tyrosine hydroxylase and dopamine-β-hydroxylase immunohistochemistry: Evidence for dopaminergic cells lying in the pons of the beef brain

Despite numerous data on the organization of the central catecholaminergic (CA) system, the nature of many CA terminal fields, fiber pathways and cell bodies, remains unknown. The discrepancies between the results obtained by CA histofluorescence and dopamine-ß-hydroxylase (DBH) immunofluorescence method suggest that certain areas described as noradrenergic (NA) may be in fact dopaminergic (DA). A method to solve this problem is to compare the localization of the two CA synthetizing enzymes: tyrosine hydroxylase (TH) and DBH. The first enzyme is a marker of all CA neurons, whereas the second one is a specific marker of noradrenergic and adrenergic neurons. We decided to develop a technique which allows visualization of both antigens on a same section. We chose to combine immunofluorescence and immunoperoxidase techniques which yield very different staining. The optimal conditions for this double labeling technique were developed in bovine brain, the homologous system for our antibodies. The results obtained by this procedure provide evidence that DA cells together with NA cells extend throughout the rostral part of the pons, and outline a successful technique for a more extensive investigation of the DA system. The same method should be applicable to other investigations involving other antigens.     

Specific localization of the guanosine triphosphate (GTP) cyclohydrolase I-immunoreactivity in the human brain

Summary. Guanosine triphosphate (GTP) cyclohydrolase I (GCH) is the first and rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase (TH). Our previous study reported the presence of GCH in several neuronal groups in animal brains using a newly raised anti-GCH antibody. The present study aims at elucidating whether GCH and TH coexist in the same neurons of the human brain with the aid of immunohistochemical dual labeling. GCH-immunoreactivity was observed in the cell bodies and fibers of monoaminergic neurons of the human brain. Neurons which contain both enzymes are seen in the human substantia nigra, ventral tegmental area, locus coeruleus, dorsal raphe, and zona incerta. In these regions, almost all the cells also show immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step enzyme for catecholamine synthesis, indicating that these neurons are catecholaminergic. However, some neurons in the dorsal and dorsomedial hypothalamic nuclei are stained only for GCH or TH. They appear to constitute an independent cell group in the human brain. The present observation suggests that L-dopa is not produced in the cells immunoreactive for TH but not for GCH, and that TH in these cells which lack GCH may have an unidentified role other than dopa synthesis. Received November 10, 1998; accepted December 22, 1998     

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.     

Vesicular and cytoplasmic localization of neurotensin-like immunoreactivity (NTLI) in neurons postsynaptic to terminals containing NTLI and/or tyrosine hydroxylase in the rat central nucleus of the amygdala.

Neurotensin and catecholamines in the central nucleus of the amygdala (CNA) have both been implicated in the integration of autonomic responses to stress. We examined whether there might be a cellular substrate for interactions involving these putative neurotransmitters in the CNA. Sections of acrolein-fixed rat brain were processed either (1) for the ultrastructural localization of a rat antiserum against neurotensin using the peroxidase-antiperoxidase (PAP) method, or (2) for the dual localization of rat neurotensin antiserum and rabbit antiserum against the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), using the PAP method and immunoautoradiography. The rat polyclonal antiserum against neurotensin was shown in immunoblots to recognize neuromedin N and Lys-Arg-neurotensin (LANT-6) in addition to neurotensin. In single and dual labeling studies, the neurotensin-like immunoreactivity (NTLI) was detected in perikarya and processes. The NTLI was localized predominantly to dense core vesicles in one group of perikarya and dendrites, while a second group had labeling both in dense core vesicles and more diffusely throughout the cytoplasm. Terminals also showed NTLI, particularly in association with dense core vesicles. The labeled terminals formed primarily symmetric junctions with both cell bodies and dendrites. In the dual labeling study, perikarya contained only NTLI while terminals contained TH and/or NTLI. Terminals containing TH or NTLI separately innervated cell bodies and dendrites displaying NTLI, and formed separate or convergent inputs onto unlabeled neuronal targets. Terminals colocalizing both TH and NTLI formed junctions only on unlabeled dendrites. These findings show that in the rat CNA two populations of neurons differ with respect to their distribution of NTLI, and that the output from neurons containing NTLI is modulated by direct synaptic input from terminals containing neurotensin and/or catecholamines. Release of neurotensin and catecholamines, most likely dopamine, from the same or separate terminals on common targets in the CNA may account for certain similarities in their stress-related functions.     

Development of tyrosine hydroxylase-immunoreactive cell populations and fiber pathways in the brain of the dogfish Scyliorhinus canicula: New perspectives on the evolution of the vertebrate catecholaminergic system

Developmental studies of the central catecholaminergic (CA) system are essential for understanding its evolution. To obtain knowledge about the CA system in chondrichthyans, an ancient gnathostome group, we used immunohistochemical techniques for detecting tyrosine hydroxylase (TH), the initial rate‐limiting enzyme of the CA synthesis, to study: 1) the neuromery of developing TH‐immunoreactive (ir) neuronal populations, 2) the development of TH‐ir innervation, and 3) the organization of TH‐ir cells and fibers in the brain of postembryonic stages of the shark Scyliorhinus canicula. The first TH‐ir cells appeared in the hypothalamus and rostral diencephalon (suprachiasmatic, posterior recess and posterior tubercle nuclei at embryonic stage 26, and dorsomedial hypothalamus at stage 28); then in more caudal basal regions of the diencephalon and rostral mesencephalon (substantia nigra/ventral tegmental area); and later on in the anterior (locus coeruleus/nucleus subcoeruleus) and posterior (vagal lobe and reticular formation) rhombencephalon. The appearance of TH‐ir cells in the telencephalon (pallium) was rather late (stage [S]31) with respect to the other TH‐ir prosencephalic populations. The first TH‐ir fibers arose from cells of the posterior tubercle (S30) and formed recognizable ascending (toward dorsal and rostral territories) and descending pathways at S31. When the second half of embryonic development started (S32), TH‐ir fibers innervated most brain areas, and nearly all TH‐ir cell groups of the postembryonic brain were already established. This study provides key information about the evolution of the developmental patterns of central CA systems in fishes and thus may help in understanding how the vertebrate CA systems have evolved. J. Comp. Neurol. 520:3574–3603, 2012. © 2012 Wiley Periodicals, Inc.     

Organization of midbrain catecholamine-containing nuclei and their projections to the striatum in the North American opossum, Didelphis virginiana.

Presumptive catecholamine (CA) neurons in the opossum midbrain were identified by tyrosine hydroxylase immunohistochemistry. In the midline, small to moderate number of CA cells were present in the rostral third of the nucleus raphe dorsalis and throughout the nucleus linearis. Ventrolaterally, such cells were observed in the deep tegmental reticular formation, in all subnuclei of the ventral tegmental area, and in the three subdivisions of the substantia nigra. The CA cells in these areas conform to the dopamine cell groups, A8, A9, and A10 as described in the rat. In several areas there appeared to be no separation between the CA neurons belonging to cytoarchitecturally different nuclei. In order to determine which CA neurons gave rise to striatal projections, the neostriatum was injected with True Blue (TB), and sections through the midbrain were processed for tyrosine hydroxylase (TH) and visualized by immunofluorescence. Neurons containing both TB and TH were observed in each of the CA cell groups mentioned above. The distribution of these cells confirmed organizational features that may be unique to the opossum's substantia nigra. In addition, different patterns of labeling resulted from caudate versus putamen injections, suggesting a rudimentary medial to lateral topography in the organization of nigrostriatal projections. Although our results suggest that the organization of midbrain CA neurons in the opossum is similar to that in placental mammals, it is clear that differences exist.     

Electron Microscopy of Colocalization of GABA and Tyrosine Hydroxylase Expression in Rat Olfactory Bulb Transplants

Juxtaglomerular (JG) neurons of rat olfactory bulb (OB) are a subset of inhibitory interneurons within the OB, acting via lateral inhibition to modulate the afferent input of the primary olfactory nerve. The JG neurons, composed of periglomerular, external tufted, and short axon cells, have been found to express various neurotransmitters, including γ-amino butyric acid (GABA) and dopamine. A specific set of neurons within the periglomerular population have also been shown to coexpress these neurotransmitters. Deafferentation or functional odor deprivation of the normal OB causes a loss of tyrosine hydroxylase (TH) (the rate limiting enzyme in the dopamine synthesis pathway) expression within the JG cell population, but appears to have no effect on GABA levels. Our laboratory has developed a transplantation model to further study the effects of deafferentation and subsequent reinnervation within this system. Sections from transplant (TX) OBs were reacted for GABA and TH using immunocytochemical localization protocols and studied by electron microscopy. Numerous neuronal populations were found to be either TH or GABA positive in this study, with a specific subpopulation showing colocalization of both. Although the architecture of the TX OB is somewhat disrupted and the TH- and GABA-positive cells were not as uniform in their arrangement as they are in the normal OB, we found that these cells in the TX OB were morphologically similar to the JG cells of normal OB. Positively labeled profiles were also found to receive and form numerous synaptic contacts with both host olfactory nerve axons as well as with the processes of donor neurons. These synaptic contacts were within areas that resemble the glomeruli of normal OB, suggesting that lateral inhibition may occur within the TX OB as it does in the normal. The coexpression of GABA and TH within specific neurons also indicates that a unique population of JG neurons that occur in normal OB are also found within this transplanted system as well.     

Ultrastructural analysis of synapses involving tyrosine hydroxylase-containing neurons in the ventral periventricular hypothalamus of the macaque

Immunocytochemical staining for tyrosine hydroxylase (TH) in the adult macaque brain revealed a network of catecholaminergic (CA) cell bodies and fibers in the arcuate (ARC), anterior ventral periventricular (APV) and lateral suprachiasmatic nuclei (SCN). Coronal Vibratome sections immunostained with PAP or colloidal gold (15 nm) were thin sectioned and examined by electron microscopy. We examined 280 TH-immunopositive processes in individual or in serial thin sections. Of these, 190 engaged in a total of 270 synapses identified as Gray Type I asymmetrical synapses (AS) with distinct postsynaptic densities or Gray Type II symmetrical synapses (SS) without such specializations. The majority (80%) of all synapses were axodendritic, 63% of which exhibited SS and 37% AS, representing almost all of the AS observed. In nearly every case, unlabeled axon terminals containing round, 45 nm, clear vesicles and occasional small dense core vesicles contacted TH-labeled dendrites. About 15% of the synapses were dendrodendritic, all of which were symmetrical. Rare contacts involving other elements (axosomatic, dendrosomatic) constituted only 5% of the total, and occurred predominantly as SS. The predominance of AS and the prevalence of SS almost exclusively on TH-containing dendrites indicates that these CA neurons receive extensive afferent input from other neurotransmitters. TH-labeling of both neural elements in most dendrodendritic, and in some axodendritic SS, also suggests that they modulate one another within the ARC, APV and SCN. The results suggest that these CA neurons perform an important role in local integration, and may act elsewhere to affect the common final pathway of the neuroendocrine system in primates.     

Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine, TPA, IBMX/forskolin), will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study, we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover, the induced TH present in these cells was enzymatically active, resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover, hNTs may serve, in vivo, as a source of human DA neurons for use in transplantation therapies.     

Expression of tyrosine hydroxylase in neurons of cultured cerebral cortex: evidence for phenotypic plasticity in neurons of the CNS

In vivo, neurons of the cerebral cortex of rat embryos did not stain with antibodies to the catecholamine (CA) biosynthetic enzyme tyrosine hydroxylase (TH) even when examined using a highly sensitive technique for radioimmunocytochemistry. However, when embryonic day (E) 13 cortex was grown 1 d in culture, several thousand cells expressed immunoreactive and catalytically active TH. All TH cells simultaneously labeled with the neuronal enzyme, neuronal specific enolase, indicating that the TH was exclusively localized in neurons. Moreover, all TH neurons were postmitotic since they did not incorporate 3H-thymidine. With time in culture, the number of TH cells selectively declined from nearly 3000 cells at 2 d to several cells at 14 d. Similarly, the number of neurons competent to express TH in culture declined with advancing age of the donor embryo. Thus, by E18, very few cortical neurons had the capacity to express TH. We conclude that during a critical period of development, postmitotic cerebral cortical neurons can express catecholamine traits in vitro but not in vivo. Thus, the neurotransmitter phenotype of certain classes of central neurons is not fixed but can be influenced by epigenetic factors found in their environment, thereby providing evidence of phenotypic plasticity in the central nervous system (CNS).     

Presynaptic transmitters and depolarizing influences regulate development of the substantia nigra in culture

Recent evidence suggests that extracellular signals regulate neurotransmitter traits in brain catecholaminergic (CA) neurons as in the periphery. Development of the dopaminergic phenotype in the mouse substantia nigra (SN) was studied by monitoring tyrosine hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis in vivo and in culture. Explants of SN were dissected from embryonic day 15 embryos and grown in culture for a week. To define the influence of depolarizing signals on central dopaminergic neurons, cultures were grown with the pharmacologic depolarizing agent veratridine. This treatment elicited a significant increase in TH enzyme activity, accompanied by elevated levels of enzyme protein. The increase in activity was prevented by TTX, suggesting that transmembrane Na+ influx was necessary for the rise in TH. A physiologic presynaptic agonist, substance P, also evoked a significant increase in TH activity; however, the coproduced tachykinin peptide, substance K (SK, neurokinin A) elicited a more dramatic rise. The SK effect was blocked by TTX, suggesting that the physiologic agonist was acting through the same mechanism as the pharmacologic agent veratridine. Immunoblot analysis revealed that SK elicited a parallel increase in TH enzyme protein. Our observations suggest that the novel peptide, SK, serves a physiological role in the regulation of TH in the striatonigral pathway.