Mesencephalic Dopamine Neurons Research Articles

The 5-HT1A receptor selective ligands, (R)-8-OH-DPAT and (S)-UH-301, differentially affect the activity of midbrain dopamine neurons

The effects of the selective 5-HT1A receptor agonist (R)-8-hydroxy-2(di-n-propylamino)tetralin [(R)-8-OH-DPAT] and the novel 5-HT1A antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)-tetralin [(S)-UH-301] were studied with regard to the firing pattern of single mesencephalic dopamine (DA) neurons with extracellular recording techniques in chloral hydrate anesthetized male rats. Neuronal activity was studied with respect to firing rate, burst firing and regularity of firing. In the ventral tegmental area (VTA) low doses of (R)-8-OH-DPAT (2-32 micrograms/kg i.v.) caused an increase in all three parameters. The effect on firing rate of DA neurons was more pronounced in the parabrachial pigmentosus nucleus than in the paranigral nucleus, the two major subdivisions of VTA. In the substantia nigra zona compacta (SN-ZC), (R)-8-OH-DPAT (2-256 micrograms/kg i.v.) had no effect on firing rate and regularity of firing and only slightly increased burst firing. High doses of (R)-8-OH-DPAT (512-1024 micrograms/kg i.v.) decreased the activity of DA cells in both areas, an effect that was prevented by pretreatment with the selective DA D2 receptor antagonist raclopride. (S)-UH-301 (100-800 micrograms/kg i.v.) decreased both firing rate and burst firing without affecting regularity of DA neurons in the VTA. In the SN-ZC, (S)-UH-301 decreased the firing rate but failed to affect burst firing and regularity of firing. These effects of (S)-UH-301 were blocked by raclopride pretreatment. Local application by pneumatic ejection of 8-OH-DPAT excited the DA cells in both the VTA and the SN-ZC, whereas (S)-UH-301 inhibited these cells when given locally. These results show that 5-HT1A receptor related compounds differentially affect the electrophysiological activity of central DA neurons. The DA receptor agonistic properties of these compound appear to contribute to the inhibitory effects of high doses of (R)-8-OH-DPAT and (S)-UH-301 on DA neuronal activity. Given the potential use of 5-HT1A receptor selective compounds in the treatment of anxiety and depression their effects on central DA systems involved in mood regulation and reward related processes are of considerable importance.

Expression of the mRNA for the 695 amino acid isoform of the β-amyloid protein precursor (β-APP 695) in ventral mesencephalic dopamine (DA) neurons of the human, monkey and rat brain

Expression of the mRNA for the 695 amino acid isoform of the β-amyloid protein precursor (β-APP 695) in ventral mesencephalic dopamine (DA) neurons of the human, monkey and rat brain

Long-term cocaine administration is not neurotoxic to cultured fetal mesencephalic dopamine neurons

The psychostimulants cocaine and methamphetamine produce their euphoric effects through an interaction with the mesolimbic dopamine system. Methamphetamine, unlike cocaine, has been shown to be neurotoxic to both dopaminergic and serotonergic systems. We have previously determined that a 6 day exposure to methamphetamine causes neuronal damage to tyrosine hydroxylase-immunopositive cells in our tissue culture model of the mesencephalon. Over the same exposure period, cocaine neither impaired neuronal function nor altered dopamine cell survival. To test whether a longer exposure period to cocaine would alter dopamine function, we added cocaine (100 μM) to the cultures once daily for either 8 or 11 days and examined changes in dopamine uptake, cell survival and morphology 24 h after the last administration. Cocaine did not produce any signs of neurotoxicity in the mesencephalic cultures.

Opposite effects of mesencephalic microinjections of cholecystokinin octapeptide and neurotensin-(1–13) on brain stimulation reward

Changes in operant responding for brain stimulation reward were studied before and after a microinjection of 1 nmol of sulphated cholecystokinin octapeptide, neurotensin or saline into the ventral tegmental area. Neurotensin produced a significant and long lasting decrease in the stimulation frequency required to produce a half-maximal rate of responding; cholecystokinin had the opposite effect, attenuating the rewarding efficacy the stimulation during the first 30 min post-injection. It is suggested that the opposite effects of the peptides on reward are due to their differential modulatory effects on a subpopulation of mesencephalic dopamine neurones.

Differential expression of acidic and basic FGF in the rat substantia nigra during development.

Both acidic (aFGF) and basic (bFGF) fibroblast growth factors have been shown to be present in the adult rat ventral mesencephalon and to exert effects on cultured mesencephalic cells. In the present study we have examined the expression of aFGF and bFGF in the rat ventral mesencephalon at various stages of development. bFGF was present at all ages examined [embryonic day 16 (E16) to postnatal day 90 (P90)]. In contrast, aFGF was not detectable at embryonic and early postnatal ages, but was observed at later (P20, P60, P90) postnatal stages. These data suggest that aFGF and bFGF may have functions in mesencephalic dopamine neurones in different stages of development.

Dopaminergic transplants in experimental parkinsonism: cellular mechanisms of graft-induced functional recovery

The ability of intrastriatal grafts of fetal mesencephalic dopamine neurons to ameliorate the symptoms of experimental and clinical parkinsonism has raised the question of the mechanisms underlying the transplant-induced functional effects. Recent studies have taken advantage of quantitative cytochemical and in situ hybridization techniques to study functional graft-host interactions at the cellular level in the rat Parkinson model. The results provide evidence that behaviorally functional grafts restore dopaminergic neurotransmission and normalize dopamine receptor function in the denervated striatum, and that these effects are likely to depend on both synaptic and extrasynaptic mechanisms.

Temporal and compartmental restriction of neuron-specific enolase expression in the rat mesostriatal system

The striatum and the mesencephalic dopamine neurons which innervate it, are each organized into developmentally and biochemically distinct compartments. Striatal patches, characterized in the neonate by high concentrations of opiate receptors and substance P, are innervated prenatally by fibers originating in one group of midbrain dopamine neurons, the ventral tier. By the third postnatal day, a dense dopamine projection from neurons in the dorsal tier of the mesostriatal group innervates non-patch areas of the striatum, i.e. the matrix, and is followed by the appearance there of neurotensin, somatostatin and calcium binding protein. We have recently observed that the period of establishment of connections between dorsal tier dopamine neurons and their target cells in the striatal matrix is accompanied by a surge in expression of the gene coding for tyrosine hydroxylase (TH). In order to determine the overall metabolic state of mesencephalic and striatal neurons during the period of up-regulation of TH gene expression, we have applied immunocytochemistry for neuron specific enolase (NSE), and cytochrome oxidase histochemistry, known markers for neuronal activity, as well as TH immunohistochemistry to the mesencephalon and striatum of postnatally developing rats. At birth, both NSE and cytochrome oxidase were expressed almost exclusively in the patches, appearing in the matrix only after the 2nd postnatal day. Patches of NSE remained visible thru the 14th day. In the mesencephalon, cytochrome oxidase and immunoreactive NSE cells in adjacent sections, were present only in the pars reticulata (i.e. ventral tier). By day 8, both techniques identified nigral cells in the dorsal as well as ventral tiers. The overlapping patterns of NSE, CO and TH distribution observed in both the striatum and mesencephalon may be related to the establishment of connections between the specific sub-populations of nigral dopamine neurons and their respective striatal target neurons.

Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture.

Dopamine (DA)-containing neurons in primary dissociated cell cultures derived from the embryonic mouse mesencephalon (day E13) were studied by histochemical and electrophysiological techniques. DA neurons exhibited two distinct morphologies, fusiform and multipolar, tended to reside in groups and organize dendrites into common fascicles. While these neurons expressed the cell-surface marker acetylcholinesterase, the presence of this enzyme could not be used to identify DA neurons unequivocally, since it was also observed in nondopaminergic cells. Neurons were therefore identified as DA by their distinct morphology, and this identification was validated with a double-labeling procedure that entailed the intracellular deposition of a fluorescent dye (Lucifer yellow or ethidium bromide), followed by processing for tyrosine hydroxylase immunocytochemistry. DA neurons identified in this manner were observed to have resting membrane potentials between -50 and -75 mV, input resistances of 50-360 M omega, and membrane time constants of 4.1-14.1 msec. Forty-seven percent of these cells displayed spontaneous activity that was irregular in nature and often contained bursts (burst length was between two and six action potentials). The DA neurons displayed a variety of ionic conductances, including (1) a Na+ conductance (gNa) that underlies the action potential, (2) Ca2+ conductances (gCa) that mediate the nonsomatic low- and high-threshold spikes observed, and (3) at least three K+ conductances (gK). Voltage-clamp analysis revealed several distinct transmembrane ionic currents, including (1) a large, rapidly inactivating tetrodotoxin-sensitive inward Na+ current (INa), (2) a 4-aminopyridine-sensitive, transient early outward K+ current that required a conditioning hyperpolarization of the membrane to be activated by a subsequent depolarization (A-current, IA), (3) a slowly developing inward current that was seen only after a conditioning hyperpolarization of the membrane and that was dependent on the presence of external Ca2+ ions (ICa), and (4) a late-onset, noninactivating K+ current. Between 25% and 54% of the late-onset K+ current was Ca(2+)-dependent and was not affected by tetraethylammonium ions. This current was termed IAHP. The remaining current was not sensitive to changes in the extracellular Ca2+ concentration but was blocked by external tetraethylammonium. This current was termed IK. The direct pressure application of DA (1-200 microM) onto the soma dose-dependently hyperpolarized these neurons; this effect was potentiated by the presence of the catecholamine reuptake blocker cocaine hydrochloride (10-200 microM). Under voltage-clamp conditions, DA was observed to increase IK significantly and had little effect on IAHP.(ABSTRACT TRUNCATED AT 400 WORDS)

Pre- and postnatal development of high-affinity [ 3H]nicotine binding sites in rat brain regions: an autoradiographic study

The ontogeny of high affinity nicotinic cholinergic binding sites was studied in Long-Evans rat brain by in vitro autoradiography, using [ 3H]nicotine (10 nM) and cold (−)nicotine bitartrate to assess specificity. The first binding sites become detectable in spinal cord and caudal medulla oblongata at gestational day (GD) 12. Until GD 14, labelling spreads throughout lower brainstem, mesencephalon and parts of diencephalon, with higher densities in ventral areas (including the area of developing mesencephalic dopamine neurons). Matrix zones remain unlabelled. Receptor sites appear in the cerebellar anlage by GD 15, and i n caudal caudate-putamen by GD 16. During development from late gestational to early postnatal stages, labelling is reduced in many lower brainstem areas and increases in forebrain, in particular in neocortex. Receptor density remains high in thalamus. In neocortex, nicotinic receptor sites are first seen in the subplate layer by GD 20. Labelling of this zone remains prominent until PN 14, when an additional band of increased receptor density is seen in cortical layers III/IV which contain high receptor levels in adulthood. At PN 27, the pattern has become similar to the adult one. The development of [ 3H]nicotine-binding sites in individual brain regions, with a general caudo-rostral gradient, accompanies cell differentiation and early synapse formation, e.g., in neocortex. The ontogenetic pattern differs in detail from that of muscarinic-cholinergic binding sites. The early presence of binding sites provides a basis for specific actions of nicotine on the fetal brain. As a consequence of the ontogenetic changes, different brain structures become targets for the action of this drug at different stages of development.

Degeneration of mesencephalic dopamine neurons in weaver mutant mice

Degeneration of mesencephalic dopamine neurons in weaver mutant mice

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.

Hypertrophy of dopamine neurons in the primate following ventromedial mesencephalic tegmentum lesion

Morphological changes in ventral mesencephalic dopamine (DA) neurons of a monkey sustaining a unilateral electrolytic lesion of the ventromedial mesencephalic tegmentum four years earlier were examined. Substantia nigra (A9) DA neurons lateral to the lesion underwent hypertrophic changes. The mean area of these neurons was enlarged by approximately 30% relative to corresponding neurons in the contralateral substantia nigra. Semi-quantitative immunohistochemical measurements of the intensity of tyrosine hydroxylase-like immunoreactivity (TH-li) indicated an increase in the amount of TH-li protein per cell in the hypertrophied neurons. Hypertrophic changes were also observed in ipsilateral A11 DA neurons of the caudal hypothalamus, suggesting that the increase in size was related to transection of the axons of DA neurons as they pass through the midbrain in their projections to target sites. The lesion did not overtly change the density or pattern of the substance P innervation of the substantia nigra, indicating that the striato- and pallido-nigral projections were spared by the lesion. These data suggest that hypertrophy may be a compensatory mechanism of dopaminergic neurons in response to partial lesions of the nigrostriatal system, and thus represent a morphological counterpart to the compensatory biochemical processes effected in response to partial lesions of the striatal dopaminergic innervation.

Burst firing of mesencephalic dopamine neurons is inhibited by somatodendritic application of kynurenate

Midbrain dopamine neurons of the zona compacta substantia nigra (SN) and ventral tegmental area (VTA), giving rise to the nigrostriatal and mesolimbocortical midbrain dopamine pathways, respectively, typically display a spontaneous activity consisting of single spikes and bursts. Previously, intracerebroventricular administration of the excitatory amino acid (EAA) antagonist kynurenate has been shown to inhibit burst firing and induce a regular, pacemaker-like firing of ventral tegmental area midbrain dopamine neurons. In the present experiments, zona compacta substantia nigra and ventral tegmental area midbrain dopamine neurons were recorded in the chloral hydrate anaesthetized male rat. Kynurenate was administered locally, either by micro-iontophoresis or by pneumatic (micropressure) ejection. Both forms of local kynurenate application produced an immediate inhibition of burst firing and a slightly increased regularity of firing in both zona compacta substantia nigra and ventral tegmental area midbrain dopamine neurons. The present results indicate that excitatory amino acid nerves tonically modulate midbrain dopamine neuronal burst firing directly on the midbrain dopamine cell bodies, further stressing the importance of excitatory amino acid innervation in the physiological function of midbrain dopamine neurons, particularly in the dynamic aspects involved in the behavioural modulation and pharmacological responses of these psychopharmacologically important neurons.

Stress Selectively Increases Fos Protein in Dopamine Neurons Innervating the Prefrontal Cortex

Stress-induced alterations in expression of c-fos protein (Fos) in mesencephalic dopamine (DA) neurons of the rat were examined in order to discern which midbrain DA neurons are metabolically activated by stress. Restraint stress for 30 min increased the number of DA neurons exhibiting Fos-like immunoreactivity in the ventral tegmental area (VTA), but not in the substantia nigra or retrorubral field. Stress elicited an increase in the number of DA neurons expressing Fos in specific nuclei within the VTA. Administration of the anxiogenic beta-carboline FG 7142 also increased the total number of VTA DA neurons expressing Fos protein, whereas pretreatment with an anxiolytic benzodiazepine (diazepam) partially prevented the stress-induced increase in Fos expression. Restraint stress for 30 min increased concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens and striatum, as well as in the prefrontal cortex. Retrograde tracer studies revealed that stress increased Fos protein expression in a distinct subset of DA neurons projecting to the prefrontal cortex. In contrast, Fos expression was not increased in any DA neurons projecting to the nucleus accumbens. The present data indicate that there are at least two functionally distinct DA systems embedded within the prefrontal cortex of the rat.

Behavioral evidence of depolarization block of mesencephalic dopamine neurons by acute haloperidol in partially 6-hydroxydopamine-lesioned rats.

The effects of acute haloperidol (HAL) and apomorphine (APO) administration were tested on the responsiveness of rats to medial forebrain bundle (MFB) brain stimulation reward (BSR) before and 28 days after partial dopamine (DA) depletion. Before 6-hydroxydopamine (6-OHDA) lesioning, HAL (50-100 micrograms/kg) and APO (25 micrograms/kg) both caused moderate decreases in the animals' responsiveness to BSR. In contrast, the same moderate doses of HAL completely abolished BSR after lesioning. However, postlesion responding for BSR after HAL could be reinstated by APO. The results of this study provide behavioral evidence that is consistent with recent electrophysiological data showing that acute HAL causes DA cells that survive 6-OHDA lesions to be driven into a state of depolarization block.

Behavioral evidence of depolarization block of mesencephalic dopamine neurons by acute haloperidol in partially 6-hydroxydopamine-lesioned rats.

Behavioral evidence of depolarization block of mesencephalic dopamine neurons by acute haloperidol in partially 6-hydroxydopamine-lesioned rats.

Enhanced survival of cultured dopamine neurons by treatment with soluble extracts from chemically deafferentiated striatum of adult rat brain

A soluble fraction was extracted from a chemically deafferentiated striatum of adult Wistar rats after unilateral lesioning of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) injection. The soluble extract from the lesioned side enhanced the survival of cultured mesencephalic dopamine (DA) neurons of 14-day-old rat embryos as evidenced by quantitative counting of tyrosine hydroxylase-like immunoreactive cells. The neurotrophic activity of this striatal extract for DA neurons was highest 14 days after 6-OHDA injection and became negligible in 28 days. The extract showed no promoting effects on cultured γ-aminobutyric acid (GABA)-containing mesencephalic neurons. These observations indicate that the striatum of adult rats may initiate de novo synthesis of trophic substance(s) for DA neurons but not for GABA neurons when subjected to nigral dopaminergic deafferentiation.

Bilateral regulation of glutamate tissue and extracellular levels in caudate-putamen by midbrain dopamine neurons

Tissue levels and in vivo release of glutamate were measured in caudate-putamen of rat brain after a unilateral 6-hydroxydopamine lesion of meso-striatal/meso-cortico-limbic dopamine neurons. The results were compared with glutamate levels from corresponding regions in sham-injected animals. The unilateral dopamine lesion induced a bilateral increase of tissue levels of glutamate (45% ipsilateral and 39% contralateral to the lesion). Extracellular levels of glutamate as measured by microdialysis were also bilaterally increased (107% ipsilateral and 94% contralateral to the lesion). The results indicate that mesencephalic dopamine neurons regulate neuronal glutamate in ipsilateral as well as contralateral caudate-putamen.

Effect of Prior Dopamine Denervation on Survival and Fiber Outgrowth from Intrastriatal Fetal Mesencephalic Grafts.

[3H]Dopamine (DA) uptake radioautography and tyrosine hydroxylase (TH) immunocytochemistry were used to assess quantitatively the effects of the presence or absence of host mesostriatal DA afferents on the survival and fiber outgrowth from fetal ventral mesencephalic DA neurons grafted into the neostriatum of adult recipient rats. Rats received bilateral intrastriatal transplants of fetal ventral mesencephalic tissue 1 month after a unilateral injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle (denervated side). Five to six months later, some of the grafted rats received a second 6-OHDA injection in the left nigrostriatal bundle (acutely denervated or 'intact' side). After a further 7 days, slices of each hemisphere from the latter rats were incubated with [3H]DA and processed for film and high resolution radioautography. The density of the film radioautographs was measured with a computerized image analysis system and calibrated by silver grain cluster (i.e. DA terminal) counting over selected areas of the same sections in light microscope radioautographs. The brains of the remaining grafted rats were processed for TH immunoreactivity 6 - 12 months after graft surgery. Neither the size of the grafts, nor the number of surviving TH-positive graft neurons showed any significant difference between the nondenervated and the denervated sides. However, the size of the TH-positive cell bodies was significantly greater in the grafts on the denervated side. In the [3H]DA uptake radioautographs, considerable outgrowth of DA fibers was evident in the neostriatum on the 'intact' side in spite of the presence of an intact host DA innervation until 7 days before sacrifice. The overall DA fiber outgrowth was nevertheless almost two-fold greater on the denervated side, and extended deeper into the host neostriatum than on the 'intact' side; only 7% of the total neostriatal area, on average, was at background level compared to 30% on the 'intact' side, and the overall density of neostriatal DA innervation amounted to 36% of normal as compared to 20% on the 'intact' side. The correlation between the overall density of graft-derived DA innervation and the size of the grafts was linear on the 'intact' side, but reached a plateau with relatively small grafts on the denervated side. However, the ventral striatum on both sides was very poorly innervated by these grafts. These findings demonstrate that the mature neostriatal tissue can support axonal growth and innervation from grafted fetal DA neurons even in the presence of a normal complement of endogenous DA fibers. Prior removal of the host striatal DA innervation does not influence the overall size of the grafts nor the number of surviving DA neurons, but induces an increase in the cell body size and fiber outgrowth of the grafted DA neurons.

Grafts of Fetal Dopamine Neurons Survive and Improve Motor Function in Parkinson's Disease

Neural transplantation can restore striatal dopaminergic neurotransmission in animal models of Parkinson's disease. It has now been shown that mesencephalic dopamine neurons, obtained from human fetuses of 8 to 9 weeks gestational age, can survive in the human brain and produce marked and sustained symptomatic relief in a patient severely affected with idiopathic Parkinson's disease. The grafts, which were implanted unilaterally into the putamen by stereotactic surgery, restored dopamine synthesis and storage in the grafted area, as assessed by positron emission tomography with 6-L-[18F]fluorodopa. This neurochemical change was accompanied by a therapeutically significant reduction in the patient's severe rigidity and bradykinesia and a marked diminuation of the fluctuations in the patient's condition during optimum medication (the "on-off" phenomenon). The clinical improvement was most marked on the side contralateral to the transplant.