Evidence for dopamine autoreceptors controlling dopamine synthesis in the substantia nigra

The effect of apomorphine and haloperidol on DOPA accumulation after inhibition of DOPA decarboxylase activity with NSD 1015 was compared in the substantia nigra (SN) and caudate nucleus (CN) of normal rats and rats deprived of nigral afferences from the striatum by means of intrastriatal kainic acid. In normal rats apomorphine decreased DOPA accumulation to the same extent in both the SN and CN. However, haloperidol produced a more pronounced increase in DOPA accumulation in the CN than in the SN. The effect of both drugs still persisted in the SN and CN after destruction of the neuronal strionigral feedback loop with kainic acid. The results provide evidence for the existence of nigral dopamine autoreceptors controlling dopamine synthesis in the SN.     

Reserpine-insensitive dopamine release in the substantia nigra?

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic pathway, but also from the dendrites in the substantia nigra (SN). Whether the DA release in the SN is sensitive to reserpine treatment, as it is in the stratum, has, however, not been clarified. We have determined the effects of reserpine on the concentrations of DA, serotonin and their metabolites in the SN and in the striatum and as an index of DA release in vivo we have assessed the accumulation of the DA metabolite 3-methoxytyramine (3-MT) following inhibition of monoamine oxidase by pargyline. The effects of reserpine on the concentrations of DA and its metabolites were different in the SN as compared to in the striatum. In the striatum there was a maximal depletion of DA to 2% of controls, but in the SN the DA concentration decreased only to 17% of controls. In the SN, the increases of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were less pronounced than in the striatum. In the striatum reserpine treatment (given 15 h, 3.75 h, or 1.75 h before pargyline) decreased the pargyline-induced 3-MT accumulation to 30% of pargyline-treated controls. However, in the SN no effects of reserpine were observed. The results indicate that DA in the SN partly is situated in a reserpine insensitive pool and that the release of DA might be insensitive to reserpine. These differences between the SN and the striatum could be due to different storage mechanisms. In the striatum DA is stored in classical storage granulas but in SN DA is partly stored in storage granulas and partly in smooth endoplasmatic reticulum.     

Evidence for autapses in the substantia nigra

Intracellular recording and intracellular HRP staining were employed to trace the recurrent terminal plexus of cat substantia nigra pars reticulata neurons. Autaptic neurons were labeled. The axon of an autaptic neuron was found to emit a recurrent collateral which distributed ‘en passage’ and terminal boutons contacting dendrites of the parent cell. Antidromic ventromedial thalamic stimulation elicited a recurrent IPSP in the autaptic neuron.     

Re-examining the ontogeny of substantia nigra dopamine neurons

Recently, the need to detail the precise ontogeny of nigrostriatal dopamine neurons has grown significantly. It is now thought that the gestational day on which the majority of these neurons are born is important not only for maximizing the yield of primary cells for transplantation but also for extracting suitable dopamine neural precursors (as stem cells) for expansion in vitro. Historically, peak ontogeny of substantia nigra pars compacta (SNc) dopamine neurons in the rat has been considered to occur around embryonic day (E)14. However, such a concept is at odds with recent studies that reveal not only that substantial numbers of tyrosine hydroxylase-immunopositive cells reside in the ventral mesencephalic region of rats at E14 but that many of these cells have matured extensive axonal projections to the ventral forebrain. Here, then, the ontogeny of SNc neurons in rats commonly used as a source of donor tissue for experimental cell transplantation in animal models of Parkinson's disease has been re-examined. Using a combination of bromodeoxyuridine (BrdU) administration at E11, E12, E13 or E14 with immunocytochemical stainings for both BrdU and tyrosine hydroxylase after 4 weeks of postnatal development, this characterization reveals that the vast majority (perhaps 80%) of SNc dopamine neurons are probably born on E12 in Sprague-Dawley rats. Such findings are important in refining the use of embryonic tissues for primary cell transplantation and may provide more precise timing for identifying the cellular and molecular events that drive neural stem cells toward a dopaminergic phenotype during development.     

Dissociation between the presynaptic dopamine-sensitive adenylate cyclase and [H]spiperone binding sites in rat substantia nigra

[3H]Spiperone binding sites and the dopamine-sensitive adenylate cyclase were measured in rat substantia nigra (s. nigra) 7 or 14 days after various lesions. Hemisections, which resulted in a 66% decline in tyrosine hydroxylase and cyclic nucleotide phosphodiesterase and a 73% decrease in glutamate decarboxylase, led to a 50% decrease in [3H]spiperone binding and to the almost complete disappearance of the dopamine-sensitive adenylate cyclase from the s. nigra on the lesioned side. 6-Hydroxydopamine injection into the s. nigra, which depleted tyrosine hydroxylase activity within the s. nigra by 85%, while leaving phosphodiesterase unaffected, resulted in a 40% decrease in [3H]spiperone binding but no change in the dopamine-sensitive adenylate cyclase. Intrastriatal injections of kainic acid did not alter tyrosine hydroxylase activity in the s. nigra, but decreased both glutamate decarboxylase (54%) and phosphodiesterase (68%); [3H]spiperone binding was unaffected by this lesion while the dopamine-sensitive adenylate cyclase was greatly reduced (50-75%). These results suggest that within the s. nigra the dopamine receptor binding sites as defined using [3H]spiperone are located on dopamine neurones while the dopamine-sensitive adenylate cyclase is located presynaptically on striatonigral nerve terminals.     

Modulation of GABA release by dopamine in the substantia nigra

The role of specific dopamine receptor subtypes in the regulation of GABA release in the substantia nigra was investigated using microdialysis in the awake rat. Both basal and potassium-stimulated changes in the extracellular concentrations of GABA were examined in response to the local perfusion of tetrodotoxin (TTX), the D1 agonist SKF 38393, or the D2 agonist LY 171555 through the microdialysis probe in the substantia nigra. Although TTX (1 microM) did not alter the basal extracellular concentrations of GABA in the substantia nigra, it attenuated the potassium-stimulated (80 mM K+) release of GABA. SKF 38393 had no effect on basal extracellular concentrations of GABA, but did potentiate K+ -stimulated release of GABA in a concentration-dependent manner. The potentiated response at the highest concentration of SKF 38393 (100 microM) was blocked by the D1 antagonist SCH 23390. In contrast to the effect of the D1 agonist, the D2 agonist LY 171555 attenuated the stimulated release of GABA. These data indicate that although basal extracellular concentrations of GABA in the substantia nigra may not be derived from neuronal pools, K+ -stimulated release of GABA is impulse-mediated and is modulated by the D1 and the D2 receptors. Local interactions between dopamine and GABA in the substantia nigra may have important implications for the direct regulation of basal ganglia efferent activity and motor behavior.     

Does cholecystokinin colocalize with dopamine in the human substantia nigra?

In situ hybridization was used to examine the distribution of neurons containing cholecystokinin (CCK) mRNA in human, monkey and rat brain. In rat and monkey brain CCK mRNA was visualized in the substantia nigra pars compacta and in the ventral tegmental area. The dopaminergic cell bodies in the human substantia nigra did not however show detectable amounts of CCK mRNA. Low levels of CCK mRNA were observed in the nucleus paranigralis, the human equivalent of the rodent ventral tegmental area. High levels of CCK mRNA were seen in other regions of the same brains including the cortex and the hippocampus. Thus, the adult human substantia nigra dopaminergic cells, in contrast to primate and rodent substantia nigra, do not express CCK. These results question the hypothesis of an involvement of CCK in the regulation of dopaminergic neurons and help to explain the absence of decreased CCK levels in the caudate and putamen of Parkinson's disease victims.     

Determination of release of endogenous dopamine from superfused substantia nigra slices.

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway but also from cell bodies and dendrites in the substantia nigra (SN). In most of the in vitro studies on DA release in the SN, release of exogenously applied 3H-DA has been determined either from slices or from synaptosomes. However 3H-DA has some disadvantages; 3H-DA is taken up and released not only from dopaminergic cells but also from other neuronal elements and radiolabelled DA may not be evenly distributed with the releasable endogenous pool of the amine. Therefore we have developed a method for determination of the release of endogenous DA from superfused guinea pig SN slices. We have used a superfusion system in which 6 slices are placed into a microchamber. Samples of superfusate were collected every 10 min for up to 3 h and biochemical analyses were performed by electrochemical detection preceded by absorption of DA on alumina and chromatography on a cation exchange column. Expected increases of the release of endogenous DA were obtained following D-amphetamine and potassium administration. The data indicate that it is possible to measure endogenous release of DA from guinea pig SN slices with standard HPLC technique and follow the release for a relatively long time.     

Immunohistochemical localization of voltage-gated calcium channels in substantia nigra dopamine neurons

The rhythmic firing of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) is thought to be mediated by nifedipine-sensitive Ca(2+) channels, although an involvement of omega-conotoxin-sensitive Ca(2+) channels is also suggested. In an attempt to localize such Ca(2+) channels at both the regional and cellular levels, their expression and distribution patterns were immunohistochemically investigated in the rat SNc. The three distinct subtypes of voltage-gated Ca(2+) channels were tested: the class B N-type alpha 1 subunit (CNB1), the class C L-type alpha 1 subunit (CNC1) and the class D L-type alpha 1 subunit (CND1). A large number of SNc neurons showed intense immunoreactivity against CND1 and they were distributed throughout the entire extent. By contrast, many fewer neurons displayed less intense CNC1 immunoreactivity and many of them were located in the lateral aspect of the SNc. No immunoreactivity against CNB1 was detected in the SNc. Moreover, double immunofluorescence analysis in combination with tyrosine hydroxylase staining revealed that virtually all DA neurons were CND1-immunoreactive whereas many DA neurons especially in the medial SNc exhibited only faint or no immunoreactivity against CNC1. Both CNC1 and CND1 were expressed in cell bodies and proximal dendrites of SNc DA neurons, whilst their distal dendrites that penetrated into the substantia nigra pars reticulata expressed CND1 alone. Thus, the ubiquitously and intensely expressed class D alpha 1 subunit of L-type Ca(2+) channels that is sensitive to both nifedipine and omega-conotoxin may be responsible for the pacemaker activity of SNc DA neurons.     

Variability in neuronal expression of dopamine receptors and transporters in the substantia nigra

Parkinson's disease (PD) patients have increased susceptibility to impulse control disorders. Recent studies have suggested that alterations in dopamine receptors in the midbrain underlie impulsive behaviors and that more impulsive individuals, including patients with PD, exhibit increased occupancy of their midbrain dopamine receptors. The cellular location of dopamine receptor subtypes and transporters within the human midbrain may therefore have important implications for the development of impulse control disorders in PD. The localization of the dopamine receptors (D1–D5) and dopamine transporter proteins in the upper brain stems of elderly adult humans (n = 8) was assessed using single immunoperoxidase and double immunofluorescence (with tyrosine hydroxylase to identify dopamine neurons). The relative amount of protein expressed in dopamine neurons from different regions was assessed by comparing their relative immunofluorescent intensities. The midbrain dopamine regions associated with impulsivity (medial nigra and ventral tegmental area [VTA]) expressed less dopamine transporter on their neurons than other midbrain dopamine regions. Medial nigral dopamine neurons expressed significantly greater amounts of D1 and D2 receptors and vesicular monoamine transporter than VTA dopamine neurons. The heterogeneous pattern of dopamine receptors and transporters in the human midbrain suggests that the effects of dopamine and dopamine agonists are likely to be nonuniform. The expression of excitatory D1 receptors on nigral dopamine neurons in midbrain regions associated with impulsivity, and their variable loss as seen in PD, may be of particular interest for impulse control. © 2013 International Parkinson and Movement Disorder Society     

Calmodulin-dependent adenylate cyclase in rat retina and the response to dopamine

Adenylate cyclase activity from the rat neural retina was highly stimulated with Ca²⁺ and calmodulin. The retinal adenylate cyclase activity was also increased by dopamine, and the activation was not changed with or without Ca²⁺-calmodulin in fractions from the neural retina homogenate after sucrose density gradient centrifugation. The results suggest that the two regulation systems (i.e. dopamine and Ca²⁺-calmodulin) of adenylate cyclase in the rat retina appear to be independent.     

Loss of Mecp2 in substantia nigra dopamine neurons compromises the nigrostriatal pathway

Mutations in the methyl-CpG-binding protein 2 (MeCP2) result in Rett syndrome (RTT), an X-linked disorder that disrupts neurodevelopment. Girls with RTT exhibit motor deficits similar to those in Parkinson's disease, suggesting defects in the nigrostriatal pathway. This study examined age-dependent changes in dopamine neurons of the substantia nigra (SN) from wild-type, presymptomatic, and symptomatic Mecp2(+/-) mice. Mecp2(+) neurons in the SN in Mecp2(+/-) mice were indistinguishable in morphology, resting conductance, and dopamine current density from neurons in wild-type mice. However, the capacitance, total dendritic length, and resting conductance of Mecp2(-) neurons were less than those of Mecp2(+) neurons as early as 4 weeks after birth, before overt symptoms. These differences were maintained throughout life. In symptomatic Mecp2(+/-) mice, the current induced by activation of D(2) dopamine autoreceptors was significantly less in Mecp2(-) neurons than in Mecp2(+) neurons, although D(2) receptor density was unaltered in Mecp2(+/-) mice. Electrochemical measurements revealed that significantly less dopamine was released after stimulation of striatum in adult Mecp2(+/-) mice compared to wild type. The decrease in size and function of Mecp2(-) neurons observed in adult Mecp2(+/-) mice was recapitulated in dopamine neurons from symptomatic Mecp2(-/y) males. These results show that mutation in Mecp2 results in cell-autonomous defects in the SN early in life and throughout adulthood. Ultimately, dysfunction in terminal dopamine release and D(2) autoreceptor-dependent currents in dopamine neurons from symptomatic females support the idea that decreased dopamine transmission due to heterogeneous Mecp2 expression contributes to the parkinsonian features of RTT in Mecp2(+/-) mice.     

Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

Quantitative autoradiography using [3H]-SCH 23390, [3H]-sulpiride and [3H]-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both [3H]-SCH 23390 and of [3H]-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). [3H]-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral [3H]-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect [3H]-SCH 23390 or [3H]-forskolin binding, but largely removed [3H]-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce [3H]-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.     

Partial depletion of dopamine in substantia nigra impairs motor performance without altering striatal dopamine neurotransmission

Previous data indicate that the release of somatodendritic dopamine in substantia nigra influences motor activity and coordination, but the relative importance of somatodendritic dopamine release vs. terminal striatal dopamine release remains to be determined. We utilized simultaneous measurement of dopamine neurotransmission by microdialysis and motor performance assessment by rotarod test to investigate the effects of local dopamine depletion in rats. The vesicular monoamine transporter inhibitor tetrabenazine (100 microm) was administered locally in substantia nigra as well as in striatum. Nigral tetrabenazine administration decreased nigral dopamine dialysate concentrations to 7% of baseline and whole-tissue dopamine content by 60%. Nigral dopamine depletion was associated with a reduction in motor performance to 73 +/- 6% of pretreatment value, but did not alter dialysate dopamine concentrations in the ipsilateral striatum. Striatal tetrabenazine administration decreased striatal dopamine dialysate concentrations to 5% of baseline and doubled the somatodendritic dopamine response to motor activity, but it was not associated with changes in motor performance or dopamine content in striatal tissue. Simultaneous treatment of substantia nigra and striatum reduced motor performance to 58 +/- 5% of the pretreatment value. The results of this study indicate that partial depletion of nigral dopamine stores can significantly impair motor functions, and that increased nigral dopamine release can counteract minor impairments of striatal dopamine transmission.     

Characterization of dopamine release in the substantia nigra by in vivo microdialysis in freely moving rats.

Dopamine (DA) is released not only from the terminals of the nigrostriatal projection, but also from the dendrites of these neurons, which arborize in the substantia nigra pars reticulata (SNR). Although striatal DA release has been extensively studied by in vivo microdialysis, dendritic DA release in the SNR has not been characterized by this technique. Extracellular DA was monitored simultaneously in the ipsilateral striatum and SNR. The nigral probe was implanted at a 50 degree angle, permitting 2.5 mm of SNR to be dialyzed. Delivery of the tracer Fluoro-Gold into the striatal probe retrogradely labeled tyrosine hydroxylase-positive cell bodies and dendrites in the vicinity of the nigral probe. Hence, it could be demonstrated that dopaminergic neurons near the nigral probe projected to the vicinity of the striatal probe. Addition of 50 mM KCl to the SNR perfusion solution produced a 3.5-fold increase in DA and a 50% reduction in dihydroxyphenylacetic acid (DOPAC) in the SNR; in contrast, this manipulation in the SNR caused DA release in the striatum to be decreased by 20%, while striatal DOPAC was increased by 50%. Local administration of nomifensine (10 microM) in the SNR produced a sevenfold increase in SNR DA but had no effect on striatal DA. Systemic injection of d-amphetamine (2 mg/kg, s.c.) elevated DA in the SNR and striatum five- to sevenfold, while DOPAC was decreased in both structures by at least 40%. To determine the effect of tetrodotoxin (TTX), basal concentrations of DA in the SNR were first elevated threefold by including nomifensine (1 microM) in the nigral perfusion solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABAB-Receptor activation alters the firing pattern of dopamine neurons in the rat substantia nigra

Previous electrophysiological experiments have emphasized the importance of the firing pattern for the functioning of midbrain dopamine (DA) neurons. In this regard, excitatory amino acid receptors appear to constitute an important modulatory control mechanism. In the present study, extracellular recording techniques were used to investigate the significance of GABA_B-receptor activation for the firing properties of DA neurons in the substantia nigra (SN) in the rat. Intravenous administration of the GABA_B-receptor agonist baclofen (1–16 mg/kg) was associated with a dose-dependent regularisation of the firing pattern, concomitant with a reduction in burst firing. At higher doses (16–32 mg/kg), the firing rate of the DA neurons was dose-dependently decreased. Also, microiontophoretic application of baclofen regularized the firing pattern of nigral DA neurons, including a reduction of burst firing. Both the regularisation of the firing pattern and inhibition of firing rate produced by systemic baclofen administration was antagonized by the GABA_B-receptor antagonist CGP 35348 (200 mg/kg, l.v.). The GABA_A-receptor agonist muscimol produced effects on the firing properties of DA neurons that were opposite to those observed following baclofen, i.e., an increase in firing rate accompanied by a Cecreased regularity. The NMDA receptor antagonist MK 801 (0.4–3.2 mg/kg, i.v.) produced a moderate, dose-dependent increase in the firing rate of the nigral DA neurons as well as a slightly regularized firing pattern. Pretreatment with MK 801 (3.2 mg/kg, i.v., 3–10 min) did neither promote nor prevent the regularisation of the firing pattern or inhibition of firing rate on the nigral DA neurons produced by baclofen. The present results clearly show that GABA_B-receptors can alter the firing pattern of nigral DA neurons, hereby counterbalancing the previously described ability of glutamate to induce burst Firing activity on these neurons. © 1993 Wiley-Liss, Inc.     

Implementation of deep neural networks to count dopamine neurons in substantia nigra

Unbiased estimates of neuron numbers within substantia nigra are crucial for experimental Parkinson's disease models and gene‐function studies. Unbiased stereological counting techniques with optical fractionation are successfully implemented, but are extremely laborious and time‐consuming. The development of neural networks and deep learning has opened a new way to teach computers to count neurons. Implementation of a programming paradigm enables a computer to learn from the data and development of an automated cell counting method. The advantages of computerized counting are reproducibility, elimination of human error and fast high‐capacity analysis. We implemented whole‐slide digital imaging and deep convolutional neural networks (CNN) to count substantia nigra dopamine neurons. We compared the results of the developed method against independent manual counting by human observers and validated the CNN algorithm against previously published data in rats and mice, where tyrosine hydroxylase (TH)‐immunoreactive neurons were counted using unbiased stereology. The developed CNN algorithm and fully cloud‐embedded Aiforia? platform provide robust and fast analysis of dopamine neurons in rat and mouse substantia nigra.     

Motor activity-induced dopamine release in the substantia nigra is regulated by muscarinic receptors

Nigro-striatal neurons release dopamine not only from their axon terminals in the striatum, but also from somata and dendrites in the substantia nigra. Somatodendritic dopamine release in the substantia nigra can facilitate motor function by mechanisms that may act independently of axon terminal dopamine release in the striatum. The dopamine neurons in the substantia nigra receive a cholinergic input from the pedunculopontine nucleus. Despite recent efforts to introduce this nucleus as a potential target for deep brain stimulation to treat motor symptoms in Parkinson's disease; and the well-known antiparkinsonian effects of anticholinergic drugs; the cholinergic influence on somatodendritic dopamine release is not well understood. The aim of this study was to investigate the possible regulation of locomotor-induced dopamine release in the substantia nigra by endogenous acetylcholine release. In intact and 6-OHDA hemi-lesioned animals alike, the muscarinic antagonist scopolamine, when perfused in the substantia nigra, amplified the locomotor-induced somatodendritic dopamine release to approximately 200% of baseline, compared to 120-130% of baseline in vehicle-treated animals. A functional importance of nigral muscarinic receptor activation was demonstrated in hemi-lesioned animals, where motor performance was significantly improved by scopolamine to 82% of pre-lesion performance, as compared to 56% in vehicle-treated controls. The results indicate that muscarinic activity in the substantia nigra is of functional importance in an animal Parkinson's disease model, and strengthen the notion that nigral dopaminergic regulation of motor activity/performance is independent of striatal dopamine release.