Mesencephalic Dopamine Neurons Research Articles

In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis

Spontaneous release and metabolism of dopamine (DA) from intrastriatal grafts of fetal mesencephalic DA neurons was measured by intracerebral dialysis. Mesencephalic DA cell suspensions were implanted into the head of the caudate-putamen in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway. Four months later, when tests for amphetamine-induced turning behaviour showed that the grafts had become functional, loops of dialysis tubing were implanted into the striatum on the grafted side and the contralateral non-lesioned side of the grafted rats, and in a similar position in the denervated caudate-putamen of 6-OHDA lesioned control rats. Dialysis perfusates collected from the 6-OHDA lesioned striata showed a reduction of about 95–98% in DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In the grafted animals these levels had recovered to about 40% of control for DA and to 12–16% of control for HVA and DOPAC. In addition, the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was increased in the grafted striata compared to both the lesioned and non-lesioned controls. Amphetamine had little or no effect on DA release in the 6-OHDA lesioned rats, but caused a marked increase in DA release in the grafted rats, this response being proportional to that seen in intact striata. Since the subsequent histochemical analysis showed that the dialysis probe had been located in the transplant-reinnervated part of the caudate-putamen, the results provide additional evidence that the grafted DA neurons exert their functional effects through a continuous active transmitter release from their newly-established terminals in the reinnervated host target.

Intrastriatal grafting of dopamine-containing neuronal cell suspensions: Effects of mixing with target or non-target cells

The effects of target and non-target cells on the growth and function of intrastriatal grafts of mesencephalic dopamine neurons have been studied in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway. Cell suspensions of ventral mesencephalon from 14–15-day-old rat fetuses (rich in developing dopamine neurons) were either grafted alone or grafted after mixing with equivalent numbers of cells obtained from the striatum (a major dopamine target area) or spinal cord (a non-target area for the mesencephalic dopamine neurons). The combined mesencephalic and striatal grafts gave rise to a greater area of dense innervation in the host caudate-putamen than grafts of mesencephalic cells alone or grafts of mesencephalic cells mixed with spinal cord cells. The number of surviving catecholamine-containing neurons did not differ significantly in the different types of grafts. In addition, there was an altered outgrowth pattern in the combined mesencephalic-striatal grafts consisting of small round islands of intensely fluorescent catecholamine-containing fibres, often in close association with the grafted dopamine neurons. In a subsequent biochemical study it was found that combined mesencephalic-striatal grafts exhibited dopamine levels and turnover that did not differ from grafts containing mesencephalic cells only. The mesencephalic-striatal cografts showed a trend toward enhanced behavioural effect, in terms of greater reduction in amphetamine-induced rotation asymmetry, when compared to other graft groups. It is suggested that the addition of embryonic striatal target cells can exert stimulatory effects on morphological development, and possibly functional parameters, of fetal dopamine cells also in vivo after intrastriatal grafting. However, the results differ in principle from those obtained in analogous in vitro studies, possibly due to the presence of adult host striatal target cells in all the groups in the present experiments.

Chapter 9 Coexistence during ontogeny and transplantation

Publisher Summary The chapter analyzes two coexistence systems—that is, the mesencephalic dopamine (DA) neurons, some of which contain cholecystokinin (CCK)-like immunoreactivity, and the 5-hydroxytryptamine (5-HT) neurons in the raphe nucleus, of which some contain SP- and/or thyrotropin-releasing hormone (TRH)-like immunoreactivity. The mesencephalic CCK neurons are transplanted either as solid grafts placed above the striatum or as cell suspensions injected directly into the brain. The chapter gives an account of the embryonic ontogeny of neuropeptide Y (NPY) and describes its relationship to the development of tyrosine hydroxylase (TH)-containing neurons in view of the coexistence of NPY and catecholamines. The chapter also discusses the relationship between TH and phenylethanolamine N-methyltransferase (PNMT)-containing neurons during fetal life. Several studies in the past with a variety of experimental models have indicated that the transplanted tissue survives and may reverse some lesion-induced, age-induced, or genetic behavioral syndromes or partly compensate specific neurochemical deficits. Recent studies on the development of different neuron populations during fetal life suggest that central neurons have certain plasticity with regard to their transmitter phenotype.

Does triiodothyronine influence the morphogenesis of fetal mouse mesencephalic dopaminergic neurons cultured in chemically defined medium?

Serum-free cultures of mouse mesencephalic cells were used for studying the influence of triiodothyronine (T3) on the morphogenesis of dopaminergic neurons. Supplementation of culture medium with T3 (10 −9 M) did not modify the number, but increased the size of the perikarya without affecting the neurite density of mesencephalic dopamine (DA) neurons. Furthermore, T3 had no effect on the uptake of [ 3H]DA in these cultures. These findings, taken together with previous results obtained with hypothalamic cell cultures, show that T3 affects the morphogenesis of mesencephalic DA neurons in a different manner than that of hypothalamus and that the pericarya size on the one hand, and the neurite elongation on the other hand, seem to be independently regulated by T3.

Cyclosporin A increases survival of cross-species intrastriatal grafts of embryonic dopamine-containing neurons.

The survival and function of cross-species (mouse-to-rat) grafts of fetal mesencephalic dopamine (DA) neurons, implanted as a cell suspension in the striatum of rats with lesions of the mesostriatal DA system, have been studied in animals with and without immunosuppression induced by Cyclosporin A (CyA). At 6 weeks after grafting 3 out of 7 non-CyA treated animals showed some degree of graft survival and variable functional compensation. In those three animals an average of 92 DA neurons per graft was counted. In the grafted animals treated with daily CyA injections, all grafts survived and produced partial or complete functional compensation, and they had an average of 557 DA neurons per graft. It is concluded that intracerebral graft survival and function can be greatly improved by CyA treatment and that the immunological protection of neural transplants in the brain is only partial.

1-methyl-4-phenylpyridine (MPP +) is toxic to mesencephalic dopamine neurons in culture

1-Methyl-4-phenylpyridine (MPP +) is the product of oxidative metabolism by monoamine oxidase of the Parkinson-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP + was tested for neurotoxicity on the dopaminergic neurons of embryonic rat midbrain in culture. Compared with MPTP, MPP + was a more potent neurotoxin for the cultured dopamine (DA) neurons as determined by decrease in [ 3H]DA uptake by the cultures and decrease in endogenous levels of DA and homovanillic acid. Catecholamine histofluorescence demonstrated the loss of fluorescing fibers after exposure to MPP +. The reduced analogue of MPTP, namely 1-methyl-4-phenylpiperidine, had no significant neurotoxic action on the dopaminergic neurons in culture.

The effects of dihydropyridines of neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of ( 3H) norepinephrine and ( 3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of ( 3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.

Dopamine and cholecystokinin immunoreactive neurones in mesencephalic grafts reinnervating the neostriatum: Evidence for selective growth regulation

Pieces of embryonic mesencephalic tissue rich in dopamine and cholecystokinin immunoreactive neurones were grafted to the dorsal surface of the caudate-putamen of adult host rats subjected to unilateral dopamine depleting lesions. After 3 months, neuronal survival in the graft and fibre outgrowth into the host brain were studied by tyrosine hydroxylase and cholecystokinin immunohistochemistry, both in serial sections and by elution and restaining of the same sections. Both dopamine- and cholecystokinin-containing neurones as well as neurones containing both compounds survived the transplantation process. The ratio of neurones in which dopamine and cholecystokinin-like immunorcactivity occurred independently and in coexistence was similar in the grafts to that seen in the intact ventral mesencephalon. This suggests that the grafted cells maintain and express at least some of their normal chemical characteristics in the ectopic cortical location. Only those fibres which contained tyrosine hydroxylase but apparently lacked the cholecystokinin-like peptide showed extensive reinnervation of the host neostriatum. The cholecystokinin-positive libres were found in a narrow zone immediately adjoining the graft. These results indicate that the dopaminergic reinnervation of the denervated neostriatum is preferentially carried out by the population of grafted mesencephalic dopamine neurones apparently lacking the cholecystokinin-like peptide. This suggests the presence of growth regulating mechanisms in the denervated neostriatum which selectively favour the ingrowth of fibres from the appropriate dopaminergic neuronal subset. The transplantation technique may therefore provide a powerful tool for the study of neurone target interactions in the establishment of neuronal connections, and of the possible role of peptidergic coexistence in the development and organization of monoaminergic pathways and their innervation patterns.

Activity of mesencephalic dopamine and non-dopamine neurons across stages of sleep and waking in the rat

Single unit activity of dopamine and non-dopamine neurons in the substantia nigra and ventral tegmental area was recorded across stages of sleep and waking in the rat. These stages consisted of slow wave sleep (SWS), rapid eye movement (REM) sleep, awake-quiet (AQ) and awake-moving (AM). The dopamine neurons showed no change in mean firing rate across the stages of sleep or waking. During REM sleep, however, the dopamine cells fired with a more variable interspike interval than during SWS. In contrast, non-dopamine neurons in the substantia nigra and ventral tegmental area showed large increases in firing rate in REM compared to SWS, and in AM compared to AQ, without showing changes in interspike interval variability. In conclusion, whereas other monoaminergic neurons and various cortical and subcortical neurons exhibit marked changes in firing rate across the stages of sleep and waking, the dopamine neurons are unique in their lack of change in firing rate across stages.

Cross-species neural grafting in a rat model of Parkinson's disease.

Previous studies1–7 have shown that intracerebral implants of embryonic mesencephalic dopamine (DA) neurones, grafted between individuals of the same inbred rat strain, can reverse some of the functional deficits caused by damage to the nigrostriatal DA pathway. These observations have raised the possibility that the intracerebral neural grafting technique may eventually find a clinical application in the treatment of neurodegenerative disorders, particularly Parkinson's disease. One obvious obstacle to any such attempts is the immunological rejection mechanisms associated with allogeneic or xenogeneic grafting. Thus, neural tissue (sensory and sympathetic ganglia), transplanted across immunological barriers to sites outside the central nervous system (CNS), are known to be rejected8,9. The brain has, however, been described as an immunologically ‘privileged’ site, partly perhaps because of its protective blood–brain barrier10–12. This may be the case for grafts of embryonic tissue, in particular. Thus, Zalewski et al.13 have reported rejection of allografts of adult ganglionic neurones transplanted o the spinal cord, while Low et al.14 found prolonged survival of embryonic brain tissue in the hippocampal region, grafted between rats of different strains. We report here long-lasting functional cross-species transplantation of mesencephalic DA neurones, taken from mouse embryos, to the dopaminergically denervated neostriatum of adult recipient rats.

Target neuron-specific process formation by embryonic mesencephalic dopamine neurons in vitro.

Mesencephalic dopamine neurons from the embryonic mouse brain were dissociated, aggregated in vitro in the presence of dissociated cells from appropriate or inappropriate target neuron areas, and visualized by the Falck-Hillarp histofluorescence technique after exposure to 1 microM exogenous dopamine. When aggregated with the surrounding rostral mesencephalic tegmentum cells only or with the addition of rostral tectum cells, the dopamine neurons formed a dense dendritic arborization, but no axons were observed. In the presence of dopamine-neuron target cells from the corpus striatum, a dense axonal plexus characteristic of that formed in this area in vivo was observed. In contrast, in aggregates formed with target cells from the frontal cortex, branching fluorescent axons bearing irregularly spaced and shaped varicosities were found coursing through the neuropil, as is characteristic of the dopaminergic innervation to the frontal cortex in vivo. Only proximal dendrites were observed in the presence of these axonal target cells. Dopamine neurons cultured with inappropriate target cells from the occipital cortex did not form either extensive axonal or dendritic processes. Thus, the presence, type, and distribution of dopamine neuronal processes are dependent on the presence of appropriate target cells. The formation of unique patterns of neuronal processes by dissociated neurons in vitro suggests that the information necessary for this differentiation is intrinsic to the dopamine neurons and their target cells. This system provides a useful model with which to study basic mechanisms underlying neuronal recognition.

A subpopulation of mesencephalic dopamine neurons projecting to limbic areas contains a cholecystokinin-like peptide: Evidence from immunohistochemistry combined with retrograde tracing

Using indirect immunofluorescence histochemistry combined with the elution and restaining technique of Tramu, Pillez & Leonardelli (1978), evidence has been obtained that at least some mesencephalic dopamine neurons contain a cholecystokinin (CCK)-like peptide, mainly the C-terminal octapeptide (CCK-8). These cholecystokinin-dopamine cells were present predominantly in the ventral tegmental area (A10), but were also found in the pars lateralis of the substantia nigra and, mainly at a rostral level, in the zona compacta. Terminal nerve fibre networks containing a cholecystokinin-like peptide and tyrosine hydroxylase (the marker for dopamine neurons), respectively, were found on adjacent sections with overlapping distribution patterns in the caudal-medial parts of the nucleus accumbens, the medial tuberculum olfactorium, the bed nucleus of the stria terminalis, the caudal periventricular caudate nucleus and the central amygdaloid nucleus. After injection of 6-hydroxy-dopamine into the lateral hypothalamus or into the ventral mesencephalon, or a frontal hemisection at the hypothalamic level, a marked reduction of both types of fibres was observed in these areas. By combining the indirect immunofluorescence technique with the retrograde tracing of fluorescent dyes, it could be established that cholecystokinin-dopamine cell bodies in the ventral tegmental area project to the caudal, medial nucleus accumbens. Colchicine injected into the lateral hypothalamus caused overlapping accumulations of cholecystokinin- and tyrosine hydroxylase-like immunoreactivity caudal to the injection site. Similar findings were seen caudal to a frontal hypothalamic hemitransection. Cholecystokinin immunoreactive neuronal structures were observed in many other brain areas and some systems of interest in relation to the ascending mesencephalic dopamine neurons are described, such as cholecystokinin fibres present in the dorsal aspects of the zona compacta and in patches in the caudate nucleus. No evidence was obtained that these two latter and some other cholecystokinin immunoreactive nerve terminal systems contained dopamine. The findings demonstrate the coexistence of a cholecystokinin-like peptide and dopamine in a population of mesencephalic neurons projecting to limbic areas.

Evidence for coexistence of dopamine and CCK in meso-limbic neurones.

Vanderhaeghen et al. reported the occurrence of gastrin-like immunoreactivity in the mammalian brain. Subsequent studies have revealed that this immunoreactivity corresponded mainly to the COOH-terminal octapeptide of cholecystokinin (CCK-8), which has a COOH-terminal pentapeptide identical to gastrin. Also, two peptides resembling the NH- and the COOH-terminal tetrapeptide fragments of CCK-8 are present in the central nervous system (CNS). Using COOH-terminal-specific antisera raised to gastrin and/or CCK, the distribution of CCK neurones has been described with immunohistochemical techniques. Although high numbers of cells and nerve terminals are found in cortical areas, the CCK systems are also present in most other parts of the brain and spinal cord. In the CNS, true gastrin molecules, gastrin-17 and gastrin-34 have been located only in the neurohypophysis, hypothalamus and occasionally in the medulla oblongata (unpublished results). We describe here the occurrence of peptides in meso-limbic dopamine neurones in the rat brain. Evidence has also been obtained that mesencephalic dopamine neurones in the human brain contain similar peptides.

The effect of morphine applied locally to mesencephalic dopamine cell bodies on spontaneous motor activity in the rat

The effect of local application of morphine (5 microgram) into the ventral tegmental area (VTA) or substantia nigra, pars compacta (SNc) on spontaneous activity was studied in the rat. Morphine in the VTA but not SNc produced an enhancement of locomotor activity which became progressively augmented with repeated injections. This effect could be blocked by systemic injections of naloxone or haloperidol. It is suggested that stimulation of opiate receptors in the vicinity of dopamine cell bodies can increase the activity of ascending mesencephalic dopamine neurones.

Response of mesencephalic and hypothalamic dopamine neurones to alpha-MSH: mediated by area postrema?

α-MELANOTROPIN (α-MSH) influences mammalian behaviour in a variety of situations. It is released from the pars intermedia of the pituitary during stress and may have a physiological role in adaptive processes1,2. In mammals, dopaminergic (DA) fibres of the tubero-hypophyseal system which originate in the arcuate nucleus of the hypothalamus, innervate the pars intermedia3; MSH secretion is usually inhibited by DA4. In rats, this DA system is subject to the influence of various extrahypothalamic brain regions thought to participate in the integration of endocrine and behavioural processes5,6. The tubero-hypophyseal DA neurones may therefore occupy a key position between brain areas involved in effects of behaviourally active peptide hormones on one hand and the pars intermedia, a site of production of such peptides, on the other hand. We report here that systemic injections of α-MSH in female rats affect the arcuate DA neurones as well as those of substantia nigra. The latter were studied in order to check for eventual parallel responses in an extrahypothalamic DA system. The peptide effect seems to depend upon the integrity of projections ascending from the lower brainstem. Cholinergic systems seem to participate in its manifestation.

Interaction of benzodiazepines with neuroleptics at central dopamine neurons.

Several benzodiazepines (chlordiazepoxide, clonazepam, diazepam and flunitrazepam) markedly counteracted the elevation of the homovanillic acid (HVA) content of the rat brain induced by neuroleptics (haloperidol, pimozide, chlorpromazine, and clozapine). A similar effect was obtained with the inhibitor of GABA transaminase, aminooxyacetic acid (AOAA). The interaction of benzodiazepines with the neuroleptic-induced HVA increase was similar in the striatum and in the limbic forebrain and was antagonized by the GABA receptor-blocking agent, picrotoxin. Both the benzodiazepines used and AOAA potentiated the cataleptic effect of the four neuroleptics. It is concluded that benzodiazepines, by intensifying GABA-ergic transmission, enhance the ongoing inhibition of mesencephalic dopamine neurons exerted by the striatonigral GABA system. As a consequence, the feedback activation of dopamine neurons induced by the neuroleptic blockade of dopamine receptors in the striatum and the limbic system is attenuated. This results in a reduction of the neuroleptic-induced increase of HVA and in the potentiation of the cataleptic effect of neuroleptics.