Different developmental potentials of diencephalic and mesencephalic dopaminergic neurons in vitro

Morphological and functional differentiation of dopamine (DA) neurons was compared in dissociated cultures from gestational day 14 rat mesencephalon and diencephalon. Numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons relative to all neurons were 4 and 1.7 times higher in mesencephalic than in diencephalic cultures at 6 and 13 days in vitro (DIV), respectively. Morphological maturation of diencephalic DA neurons was retarded in comparison to mesencephalic DA neurons. There were gross differences in soma size and length of processes between the two types of DA neurons, the appearance of which was strongly reminiscent of DA cell types described in vivo. Functional maturation of DA neurons was quantified by measuring uptake and Ca2+-dependent K+-stimulated release of [3H]DA per TH-IR neuron. As early as 6 DIV, DA uptake by mesencephalic DA neurons was saturable, was sensitive to benztropine and reserpine, and could be displaced by unlabeled DA. Twenty to 30% of the radioactivity accumulated could be released upon depolarization within a period of 5 min. At 6 DIV, influx of [3H]DA into diencephalic DA neurons was almost insensitive to benztropine, reserpine and unlabeled DA. Even after 13 DIV, diencephalic DA uptake was characterized by a markedly lower initial velocity, a longer time needed to reach saturation, a lower uptake capacity, and a lower sensitivity to benztropine than mesencephalic DA uptake. The releasable pool was very small and did not increase between DIV 6 and 13. The results demonstrate that mesencephalic DA neurons in vitro differentiate considerably faster than diencephalic DA neurons and gain functional competence very early in brain development. Comparison with data on adult nigrostriatal and hypothalamic DA systems suggests that the in vitro differences reflect a fundamental regional diversity of DA neurons.     

Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids.

Sexual differentiation of dopaminergic neurons was studied in gender-specific cultures. Dissociated cell cultures were prepared from di- or mesencephalon of gestational day 14 rat embryos and raised in the absence or presence of 17 beta-estradiol or testosterone for up to 13 days in vitro (DIV). Developmental profiles of levels of dopamine (DA) and metabolites as well as capacity for vesicular storage of the transmitter were determined by HPLC. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted. Higher levels of DA were measured in female than in male cultures of both brain regions. In mesencephalic cultures, the differences in DA levels were fully accounted for by sex differences in numbers of TH-IR cells, whereas no sex differences in cell numbers were found in diencephalic cultures. Dihydroxyphenylacetic acid (DOPAC) levels and vesicular storage capacity matured faster in mesencephalic than in diencephalic cultures, but no sex differences were observed. Homovanillic acid (HVA) could not be detected except in 13-DIV mesencephalic cultures. Hormonal treatment did not erase sexual differentiation of dopaminergic neurons. Irrespective of the gender, however, both steroids decreased DA and DOPAC contents in diencephalic cultures but not in mesencephalic cultures. It is proposed that sexual differentiation of dopaminergic systems proceeds in a region-specific fashion and that neurogenesis and development of various parameters of dopaminergic activity may be differentially affected. Sexual differentiation of dopaminergic neurons may be initiated independently of the action of gonadal steroid hormones and may subsequently be modified by differences in hormonal environment.     

Ontogeny of the metencephalic, mesencephalic and diencephalic content of catecholamines as measured by high performance liquid chromatography with electrochemical detection

Developmental changes in norepinephrine (NE), dopamine (DA) and epinephrine (E) contents of the rat metencephalon, mesencephalon and diencephalon, have been measured by high performance liquid chromatography with electrochemical detection, from fetal stages (E15 or E17 to E21) to postnatal days (P0 to P30) and compared to the adult levels. The data show a biphasic pattern in NE changes of the three brain areas, with a first increase in the late prenatal period, followed by a further development from P0 to P18, thus reaching the adult levels. A similar pattern of development is found for the mesencephalic and diencephalic DA contents. The E levels of the diencephalon are very low in comparison to the NE and DA concentrations, but present a gradual increase from E17 to P18. The results correlate with the development of catecholamine systems in brain area as measured by other methodological approaches.     

Serotonin promotes region-specific glial influences on cultured serotonin and dopamine neurons.

To test the hypothesis that glia mediate interactions between embryonic serotonergic (5-HT) neurons and dopamine neurons, we studied the effects of 5-HT in co-cultures of E14 raphe neurons of mesencephalic dopamine neurons and radial glia/astrocytes derived from the same (homotypic) or opposite (heterotypic) brain region using a dose (10(-5) M) that would produce 5-HT uptake into glial cells as well as activate 5-HT receptors. Morphometric analysis of 5-HT and tyrosine hydroxylase (TH) immunoreactive neurons revealed regional differences in the effects of 5-HT (and nialamide) on survival, cell soma size, and dendrite-like neurite outgrowth in neuronal-glial co-cultures. In general, 5-HT had more significant effects on both types of monoamine neuron when they were cultured with mesencephalic glia (GSN). Stimulatory effects of 5-HT on growth of TH neurons in GSN cultures suggest that developing raphe axons, which reach the mesencephalon during the early differentiation of these neurons, may enhance the influence of local glial-derived trophic factors. Likewise, the promotion of 5-HT neuronal survival in these cultures suggests that glial factors in the mesencephalon may contribute to the support of 5-HT neurons in addition to the influences of raphe glia. The inhibitory effects of 5-HT on neurite outgrowth by raphe neurons in GSN co-cultures indicates enhanced sensitivity of these neurons to the inhibitory effects of 5-HT in the presence of mesencephalic glia. The region-specific effects of 5-HT and nialamide in glial co-cultures suggest that raphe and mesencephalic glia may express different capacities for 5-HT uptake, receptors, and/or monoamine oxidase (MAO) activities. These characteristics could be important for the specificity of growth-regulatory influences of glial cells on the development of brain monoamine neurons.     

Prenatal ontogenesis of pro-opiomelanocortin in the mouse central nervous system and pituitary gland: an in situ hybridization and immunocytochemical study

Pro-opiomelanocortin (POMC) mRNA detected by in situ hybridization and POMC/ACTH (adrenocorticotropin)-containing neurons detected by immunocytochemistry were first observed in the presumptive arcuate nucleus of embryonic mouse brain on gestational day 10.5 (E10.5). Immunostained fibers were also evident on E10.5 in the lateral and dorsal diencephalon. In these areas, a dense network of processes developed by E11.5 and extended into the mesencephalon. Fibers were detected in the myelencephalon at this stage and a day later (E12.5) in the spinal cord. Adult-like patterns of POMC/ACTH fibers were established in the diencephalon, mesencephalon, metencephalon and the myelencephalon between E13.5 and E15.5. POMC-expressing cells in the anterior and intermediate lobes of the pituitary gland appeared on E12.5 and E14.5, respectively. The early expression of POMC and the rapid establishment of dense fiber tracts in the brain is consistent with a role for POMC-derived peptides in the development of the central nervous system.     

Mesencephalic dopaminergic cells exhibit increased density of neural cell adhesion molecule and polysialic acid during development.

In the developing mesencephalon of the rat, the dopaminergic neurons are generated in the ventricular zone of the basal plate between E11 and E15 and then migrate along radial glia to the ventral surface of the developing mesencephalon. To study the factors that control migration and maturation of the dopaminergic neurons, we immunolabeled embryo and pups, ages E12-P21, for neural cell adhesion molecule (NCAM), polysialic acid (PSA) - a polysaccharide found in high amounts on NCAM during development, tyrosine hydroxylase (TH) - a marker of mesencephalic dopaminergic cells, and vimentin - the major cytoskeletal protein in radial glia in the rat. At E13, we noted that cells throughout the mesencephalon contained NCAM-immunoreactive (NCAM-IR) material but that cells along the ventral surface of the mesencephalon contained an increased amount of NCAM-IR material and PSA-immunoreactive (PSA-IR) material. At this age, we first noted a small number of TH-immunoreactive (TH-IR) cells adjacent to the marginal zone of the ventral surface of the mesencephalon. Many of the TH-IR cells contained an increased density of NCAM-IR material. At age E14, the pattern of increased density of NCAM-IR material on cells along the ventral surface of the mesencephalon persisted and a conspicuous amount of PSA-IR material was also noted on cells in this region. TH-IR cells were more numerous, and a striking number of the TH-IR cells also contained an increased amount of NCAM-IR material and PSA-IR material. With increasing age the distribution of NCAM-IR material and PSA-IR material in the mesencephalon became more uniform. Our work suggests that NCAM may be involved in control of migration and synthesis of TH in the dopaminergic cells of the developing mesencephalon.     

Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: a whole-mount immunocytochemical study

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).     

Development of the serotonergic system in the rat embryo: An immunocytochemical study

The development of central serotonergic neurons has been examined immunocytochemically utilizing an antiserum to serotonin (5-HT). Cells of the B4-B9 complex are first detected early on embryonic day 13 (E13; 7 mm crown rump length, CRL) and increase rapidly in number through E15 when they appear as bilateral columns situated from just caudal to the mesencephalic flexure to the pontine flexure. Aggregation of cells into subgroups is apparent soon after 5-HT neurons leave the ventricular zone, allowing the identification of certain subdivisions of the B4-B9 complex long before they assume their adult locations. The initial detection of 5-HT immunoreactive cells in the medulla occurs 1-2 days after the appearance of cells in the B4-B9 complex, although it has been reported that the time of origin of medullary raphe neurons (B1-B3) occurs before that of raphe neurons in the midbrain and pons (B4-B9). The first medullary 5-HT neurons, comprising the B3 subdivision occur ventro-laterally on E14 (10-11 mm CRL) at least 1-2 days before midline 5-HT neurons are visualized in the B1 and B2 groups. Thus, in contrast to cells in the B4-B9 complex, medullary 5-HT neurons complete much of their migration before they can be detected immunocytochemically, indicating that the time of onset of transmitter synthesis and storage may differ during differentiation of cells sharing a common neurotransmitter phenotype. The formation of ascending 5-HT fiber projections occurs rapidly from cells of the B4-B9 complex. Within 24 hours after the initial detection of 5-HT fiber immunoreactivity in such cells at E13, their axons are seen entering the caudal diencephalon (E14). These fibers have traversed the diencephalon and floor of the telencephalon by E15-E16 and reach the frontal neocortical pole by E17. The main ascending bundle of 5-HT axons courses through the diencephalon in the vicinity of the medial forebrain bundle, although some fibers also diverge and travel along certain pre-existing non-5HT pathways. However, examples are also found of acute directional changes in 5-HT fiber growth which do not appear to be associated with pre-formed non-5HT pathways. The pattern of ascending fiber outgrowth suggests a priority routing system which provides certain regions with 5-HT axons in a preferential sequence irrespective of the distance of these areas from 5-HT cell groups or from major bundles of ascending 5-HT fibers.     

Effects of coculture on the morphology of identified raphe and substantia nigra neurons from the embryonic rat brain

The substantia nigra (SN) is one of the earliest targets of the 5-HT neurons of the raphe nuclei (RN). To test the hypothesis that embryonic 5-HT and catecholamine neurons may influence the differentiation of their target cells or source neurons, we have produced dissociated cell cultures from embryonic day 14 (E14) rat rhombencephalon (containing the serotonergic RN) and mesencephalon (containing the dopaminergic substantia nigra, SN). These cells were grown for 6 days in vitro, either as single cultures (RN or SN) or cocultures (RN + SN). Effects of coculture on the morphological development of neurons immunoreactive (IR) for 5-HT or tyrosine hydroxylase (TH) were studied by measuring a series of morphological parameters related to size of the cell body and dendritic field, as well as to the complexity of neurites within this field, using computer-assisted morphometry. Increases in a number of these parameters were found in cocultures compared to single cultures for both types of monoamine neurons, but a greater number of parameters were increased for TH-IR cells, including size of the cell body. Although this might suggest that there was a greater effect of coculture on the TH-IR (dopaminergic) cells of the SN than on the 5-HT-IR cells of the RN, we must consider the fact that a significant population of TH-IR cells were present in single RN cultures, which contributed to the total population of TH-IR cells in cocultures. Indeed, when morphometric parameters for TH-IR cells in RN and SN single cultures were compared, it was found that TH-IR cells from the RN were generally larger and more complex than those from the SN. Therefore, an analysis was made of which parameters were significantly increased for TH-IR cells in cocultures compared to single cultures from both SN and RN. This was the case for two parameters: cell body size and absolute field area, indicating that these increases were probably due to the effects of coculture itself rather than to contamination by the larger and more complex TH-IR cells from the RN. It is impossible to ascertain, however, whether this effect was on cells from the RN, SN, or both. Coculture effects on 5-HT-IR cells were easier to analyze, since no such cells were found in single cultures of SN.(ABSTRACT TRUNCATED AT 400 WORDS)     

The neurotrophic effects of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia

Neurotrophic support is generally believed to result from a direct action of growth factors on developing neurons. However, there is increasing evidence that growth factors can indirectly affect neuronal development by glial-mediated processes. To investigate a possible role of glia in mediating neurotrophic effects on dopaminergic neurons, four purified growth factors were screened for dual effects on the survival and differentiation of dopaminergic neurons and on the proliferation of mesencephalic glial cells in vitro. Dissociated embryonic day 14.5 rat mesencephalon was grown at low cell density without serum, conditions under which both glial growth and neuronal survival are not optimal. Treatment of these cultures with acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) increased the number of surviving tyrosine hydroxylase-immunoreactive (TH-IR) neurons by 90-110% [corrected] at 8 d in vitro in a dose-dependent manner. The effects of these factors were not additive. High-affinity dopamine uptake was increased by bFGF, but not by aFGF. Length of TH-IR neurites was not affected by either aFGF or bFGF. Both growth factors induced proliferation of mesencephalic astrocytes as demonstrated by autoradiographic labeling with 3H-thymidine combined with immunocytochemistry for glial fibrillary acidic protein (GFAP). In contrast, platelet-derived growth factor (PDGF) and interleukin-1 had no effect on the survival or differentiation of dopaminergic neurons or the proliferation of mesencephalic astrocytes. Inhibition of glial proliferation abolished the neurotrophic effects exerted by aFGF or bFGF on dopaminergic neurons. Moreover, conditioned medium derived from mesencephalic glial cultures replated in the virtual absence of neurons also contained neurotrophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and behavioral effects of serotonin neurotoxins on the nigrostriatal dopamine system: Comparison of injection sites

Unilateral injections of the serotonin neurotoxin, 5,7-dihydroxytryptamine (DHT), at various points along the 5-HT pathway to the forebrain produce a turning syndrome associated with alterations of dopamine synthesis in the ipsilateral striatum. Unilateral injections of DHT into the SN produced an ipsilateral increase in striatal dopamine (DA) turnover and contralateral rotation in response to amphetamine or apomorphine. Injection of DHT into the MFB produced an ipsilateral decrease in striatal DA turnover and tyrosine hydroxylase (TOH) activity, and ipsilateral rotation in response to amphetamine or apomorphine. After the injection of DHT into the SN or MFB, there was a significant correlation between the rates of drug-induced rotation, the decrease in cortical 5-HT turnover, and the change in striatal DA turnover, suggesting that the unilateral change in DA turnover (and, presumably, the increased stimulation of DA receptors) is causally linked to turning. Injection of DHT into the zones of the striatum and GP richest in 5-HT terminals produced the same responses as the MFB-lesioned rats: ipsilateral rotation and a decrease in striatal TOH activity. Injection of DHT into the area of the striatum richest in DA terminals failed to produce rotation or a significant change in TOH activity. We suggest that 5-HT neurons from the raphe nuclei exert a tonic inhibition on the nigrostriatal pathway at the level of the SN through direct synapses on DA neurons, whereas their neostriatal terminals have an indirect effect on DA terminals, perhaps via interaction with cholinergic and GABA-ergic neurons.     

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.     

Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia.

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.     

Dorsal raphe stimulation differentially modulates dopaminergic neurons in the ventral tegmental area and substantia nigra

The serotoninergic (5-HT) input from the dorsal raphe nucleus (DRN) to midbrain dopamine (DA) neurons is one of the most prominent. In this study, using standard extracellular single cell recording techniques we investigated the effects of electrical stimulation of the DRN on the spontaneous activity of substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) DA neurons in anesthetized rats. Poststimulus time histograms (PSTH) revealed two different types of response in both SNpc and VTA. Some cells exhibited an inhibition-excitation response while in other DA neurons the initial response was an excitation followed by an inhibition. In SNpc, 56% of the DA cells recorded were initially inhibited and 31% of the DA cells were initially excited. In contrast, 63% of VTA DA cells were initially excited and 34% were initially inhibited. Depletion of endogenous 5-HT by the neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), and the 5-HT synthesis inhibitor para-chlorophenylalanine (PCPA), almost completely eliminated the inhibition-excitation response in both SNpc and VTA DA cells, without changing the percentage of DA cells initially excited. Consequently, the proportion of DA neurons that were not affected by DR stimulation increased after 5-HT depletion (from 13% to 60% in SNpc and from 6% to 31% in VTA). In several DA cells, DRN stimulation caused important changes in firing rate and firing pattern. These data strongly suggest that the 5-HT input from the DRN is mainly inhibitory. It also suggests that 5-HT afferences modulate SNpc and VTA DA neurons in an opposite manner. Our results also suggest that non-5-HT inputs from DR can also modulate mesencephalic DA neurons. A differential modulation of VTA and SNpc DA neurons by 5-HT afferences from the DRN could have important implications for the development of drugs to treat schizophrenia or other neurologic and psychiatric diseases in which DA neurons are involved.     

Sex differences in densities of dopaminergic fibers and GABAergic neurons in the prenatal rat striatum.

On the basis of observations on dopaminergic neurons developing in gender-specific cultures of embryonic rat mesencephalon, we have hypothesized that as yet unknown sexual dimorphisms might be found in projection areas of dopaminergic neurons. Therefore we searched for possible sex differences in the striatum during the period when massive ingrowth of mesencephalic afferents occurs and the striatal gamma-aminobutyric acid (GABA)ergic neurons differentiate. Male and female rats of embryonic days (E) 16, 18, 20, and 21 were fixed by perfusion through the heart. Vibratome sections were cut from the striatal anlage and sequentially immunostained for GABA by the immunogold-silver technique and tyrosine hydroxylase (TH) by the avidin-biotin-peroxidase method. Ultrathin sections were scanned for numbers of GABA- and TH-immunoreactive (IR) elements. Densities of TH-IR axons as well as of GABA-IR cell body profiles progressed with time. Contacts between TH-IR axons and GABA-IR and immunonegative cells were observed as early as E-16, increasing in numbers toward later stages. Throughout prenatal development, female striata displayed higher densities of both TH-IR axon and GABA-IR cell body profiles than male ones. This is the first report of a distinct anatomical sex difference regarding two major components of a key center of motor control. Prenatal sexual differentiation of the striatum may lead to a sexually dimorphic extrapyramidal circuitry, the existence of which, in the adult, is suggested by experimental and clinical data.     

Sex differences in [123I]beta-CIT SPECT measures of dopamine and serotonin transporter availability in healthy smokers and nonsmokers

Nicotine and other constituents of tobacco smoke elevate dopamine (DA) and serotonin (5-HT) levels in brain and may cause homeostatic adaptations in DA and 5-HT transporters. Since sex steroids alter DA and 5-HT transporter expression, the effects of smoking on DA and 5-HT transporter availability may differ between sexes. In the present study, DA and 5-HT transporter availabilities were quantitated using single photon emission computed tomography (SPECT) imaging approximately 22 h after bolus administration of [123I]beta-CIT, an analog of cocaine which labels DA and 5-HT transporters. Forty-two subjects including 21 pairs of age-, race-, and gender-matched healthy smokers and nonsmokers (12 female and 9 male pairs) were imaged. Regional uptake was assessed by the outcome measures, V3", which is the ratio of specific (i.e., ROI-cerebellar activity) to nondisplaceable (cerebellar) activity, and V3, the ratio of specific to free plasma parent. Overall, striatal and diencephalic [123I]beta-CIT uptake was not altered by smoking, whereas brainstem [123I]beta-CIT uptake was modestly higher (10%) in smokers vs. nonsmokers. When subgrouped by sex, regardless of smoking status, [123I]beta-CIT uptake was higher in the striatum (10%), diencephalon (15%), and brainstem (15%) in females vs. males. The sex*smoking interaction was not significant in the striatum, diencephalon, or brainstem, despite the observation of 20% higher brainstem [123I]beta-CIT uptake in male smokers vs. nonsmokers and less than a 5% difference between female smokers and nonsmokers. The results demonstrate higher DA and 5-HT transporter availability in females vs. males and no overall effect of smoking with the exception of a modest elevation in brainstem 5-HT transporters in male smokers. Although these findings are preliminary and need validation with a more selective 5-HT transporter radiotracer, the results suggest that brainstem 5-HT transporters may be regulated by smoking in a sex-specific manner.     

Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain.

The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.     

Mouse epidermal growth factor-responsive neural precursor cells increase the survival and functional capacity of embryonic rat dopamine neurons in vitro.

We have grown expanded populations of epidermal growth factor (EGF)-responsive mouse striatal precursor cells and subsequently co-cultured these with primary E14 rat ventral mesencephalon. The aim of these experiments was to induce dopaminergic (DA) neuronal phenotypes from the murine precursors. While no precursor cell-derived neurons were induced to express tyrosine hydroxylase (TH), there was a dramatic 30-fold increase in the survival of rat-derived TH-positive neurons in the co-cultures. The effect was not explicable solely in terms of total plating density, and was accompanied by a significantly enhanced capacity for [3H]dopamine uptake in the co-cultures compared to rat alone cultures. The present data show that, although primary rat E14 mesencephalic cells are incapable of inducing the development of DA neurons from EGF-responsive mouse neural precursor cells, such precursors will differentiate into cells capable of enhancing the survival and overall functional efficacy of primary embryonic dopamine neurons.     

Neurturin enhances the survival of intrastriatal fetal dopaminergic transplants.

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.     

Trophic and tropic effects of striatal astrocytes on cografted mesencephalic dopamine neurons and their axons

Astrocytes from the ventral mesencephalon and from the striatum respectively promote the dendritic and axonal arborization of dopamine (DA) neurons in vitro. To test this response in vivo, astrocytes in primary cultures from the neonatal cerebral cortex, ventral mesencephalon, or striatum were coimplanted with fetal ventral mesencephalic tissue into the intact or DA-denervated striatum of adult rats and these cografts examined after 3–6 months by tyrosine hydroxylase (TH) immunohistochemistry (intact recipients) or after 5–6 months by in vitro [H]DA-uptake autoradiography (DA-denervated recipients). In contrast with single ventral mesencephalic grafts, all types of cograft displayed a rather uniform distribution of TH-immunoreactive perikarya. The average size of TH-immunoreactive cell bodies was not significantly different in cografts containing cortical or mesencephalic astrocytes and in single ventral mesencephalic grafts, but it was significantly larger in cografts containing striatal astrocytes. Nevertheless, the number of [H]DA-labeled terminals in the DA-lesioned host striatum was clearly smaller with cografts of striatal astrocytes than with single mesencephalic grafts or with cografts containing cortical astrocytes. On the other hand, cografts of striatal astrocytes contained much higher numbers of [H]DA-labeled terminals than the other types of graft or cograft. Thus, while cografted astrocytes in general influence the distribution of DA neurons within the graft, astrocytes from the neonatal striatum have a trophic effect on DA perikarya and a tropic effect on DA axons, keeping the latter within the graft.