Substantia Nigra Pars Compacta Of Mice Research Articles

Age-related changes in expression of lysine48 and lysine63 ubiquitin linkages in dopamine neurons of midbrain in mice.

Ubiquitination of target proteins is mediated via different ubiquitin lysine (K) linkages and determines the protein fates. In particular, K48 ubiquitin linkage targets proteins for degradation, whereas K63 ubiquitin linkage plays a nondegradative role. Parkinson's disease is an age-onset neurodegenerative disorder, which shows selective loss of dopamine neurons in substantia nigra pars compacta (SNC) and ubiquitinated protein aggregates. However, age-related expression of K48 and K63 ubiquitin linkages in SNC dopamine neurons remains elusive. We thus sought to explore the expression of K48 and K63 ubiquitin linkages in dopamine neurons in SNCs of mice at different ages with morphological and biochemical assays. Here our results indicated that in 5-week-old mice, dopamine neurons presented higher levels of K48 and K63 ubiquitin linkages than nondopamine neural cells. Aging promoted the formation of protein aggregates that are positive for both K48 and K63 ubiquitin linkages, together with tyrosine hydroxylase, a dopamine neuron marker. Moreover, 21-month-old mice showed fewer neural cells and tyrosine hydroxylase positive neurons in the SNCs than younger mice. Through biochemical analysis, the 21-month-old mice were shown to express more K48 ubiquitin linkages and less tyrosine hydroxylase and NeuN than the 5-week-old mice. These results suggest the first time that expression of K48 and K63 ubiquitin lysine linkages in midbrain dopamine neurons is age-related and may be involved in the loss of dopamine neurons.

In Vivo Assessment of Cell Death and Nigrostriatal Pathway Integrity Following Continuous Expression of C3 Transferase

The bacterial exoenzyme C3 transferase (C3) irreversibly inhibits RhoA GTPase leading to stimulation of axonal outgrowth in injured neurons. C3 has been used successfully in models of neurotrauma and shows promise as an option to support cell survival and axonal growth of dopaminergic (DA) neurons in Parkinson’s disease (PD) cell therapy. Whether the continuous expression of C3 in DA neurons is well-tolerated is unknown. To assess the potential neurotoxicity of sustained expression of C3 in DA neurons, we generated Cre recombinase-dependent adeno-associated viral vectors (AAV) for targeted C3 delivery to DA neurons of the mouse substantia nigra pars compacta (SNc). The effect of continuous expression of C3 on DA neurons was assessed by immunohistochemistry and compared to that of Enhanced Yellow Fluorescent Protein (EYFP) as negative controls. We did not find significant reduction of tyrosine hydroxylase (TH) expression levels nor the presence of cleaved activated caspase 3. Astrocytic activation as determined by GFAP expression was comparable to EYFP controls. To evaluate the impact of C3 expression on striatal terminals of the nigrostriatal pathway, we compared the rotational behavior of wildtype mice injected unilaterally with either C3 or 6-hydroxydopamine (6-OHDA). Mice injected with C3 exhibited similar ipsiversive rotations to the site of injection in comparison to control mice injected with EYFP and significantly fewer ipsiversive rotations compared to 6-OHDA lesioned mice. Non-significant difference between C3 and EYFP controls in behavioral and histological analyses demonstrate that transduced DA neurons express C3 continuously without apparent adverse effects, supporting the use of C3 in efficacy studies targeting DA neurons.

RGS Proteins as Critical Regulators of Motor Function and Their Implications in Parkinson's Disease.

Parkinson disease (PD) is a devastating, largely nonfamilial, age-related disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Release of DA from these neurons into the dorsal striatum is crucial for regulating movement and their loss causes PD. Unfortunately, the mechanisms underlying SNc neurodegeneration remain unclear, and currently there is no cure for PD, only symptomatic treatments. Recently, several regulator of G protein signaling (RGS) proteins have emerged as critical modulators of PD pathogenesis and/or motor dysfunction and dyskinesia: RGSs 4, 6, 9, and 10. Striatal RGS4 has been shown to exacerbate motor symptoms of DA loss by suppressing M4-autoreceptor-Gα i/o signaling in striatal cholinergic interneurons. RGS6 and RGS9 are key regulators of D2R-Gα i/o signaling in SNc DA neurons and striatal medium spiny neurons, respectively. RGS6, expressed in human and mouse SNc DA neurons, suppresses characteristic PD hallmarks in aged mice, including SNc DA neuron loss, motor deficits, and α-synuclein accumulation. After DA depletion, RGS9 (through its inhibition of medium spiny neuron D2R signaling) suppresses motor dysfunction induced by L-DOPA or D2R-selective agonists. RGS10 is highly expressed in microglia, the brain's resident immune cells. Within the SNc, RGS10 may promote DA neuron survival through the upregulation of prosurvival genes and inhibition of microglial inflammatory factor expression. Thus, RGSs 4, 6, 9, and 10 are critical modulators of cell signaling pathways that promote SNc DA neuron survival and/or proper motor control. Accordingly, these RGS proteins represent novel therapeutic targets for the treatment of PD pathology. SIGNIFICANCE STATEMENT: Parkinson disease (PD), the most common movement disorder, is a progressive neurodegenerative disease characterized by substantia nigra pars compacta (SNc) dopamine (DA) neuron loss and subsequent motor deficits. Current PD therapies only target disease motor symptomology and are fraught with side effects. Therefore, researchers have begun to explore alternative therapeutic options. Regulator of G protein signaling (RGS) proteins, whether primarily expressed in SNc DA neurons (RGS6), striatal neurons (RGSs 4 and 9), or microglia (RGS10), modulate key signaling pathways important for SNc DA neuron survival and/or proper motor control. As such, RGS proteins represent novel therapeutic targets in PD.

Cicadidae Periostracum, the Cast-Off Skin of Cicada, Protects Dopaminergic Neurons in a Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic neuronal loss in the substantia nigra pars compacta (SNPC) and the striatum. Nuclear receptor-related 1 protein (Nurr1) is a nuclear hormone receptor implicated in limiting mitochondrial dysfunction, apoptosis, and inflammation in the central nervous system and protecting dopaminergic neurons and a promising therapeutic target for PD. Cicadidae Periostracum (CP), the cast-off skin of Cryptotympana pustulata Fabricius, has been used in traditional medicine for its many clinical pharmacological effects, including the treatment of psychological symptoms in PD. However, scientific evidence for the use of CP in neurodegenerative diseases, including PD, is lacking. Here, we investigated the protective effects of CP on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced PD in mice and explored the underlying mechanisms of action, focusing on Nurr1. CP increased the expression levels of Nurr1, tyrosine hydroxylase, DOPA decarboxylase, dopamine transporter, and vesicular monoamine transporter 2 via extracellular signal-regulated kinase phosphorylation in differentiated PC12 cells and the mouse SNPC. In MPTP-induced PD, CP promoted recovery from movement impairments. CP prevented dopamine depletion and protected against dopaminergic neuronal degradation via mitochondria-mediated apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X, cytochrome c, and cleaved caspase-9 and caspase-3 by inhibiting MPTP-induced neuroinflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase 2, and glial/microglial activation. Moreover, CP inhibited lipopolysaccharide-induced neuroinflammatory cytokines and response levels and glial/microglial activation in BV2 microglia and the mouse brain. Our findings suggest that CP might contribute to neuroprotective signaling by regulating neurotrophic factors primarily via Nurr1 signaling, neuroinflammation, and mitochondria-mediated apoptosis.

Nanoscale Imaging of Synaptic Connections with Expansion Microscopy

Biologists have long looked for ways to circumvent the physical diffraction limit of light and have developed many strategies to accomplish this. While many techniques employed to image sub-diffraction-limit structures rely on sophisticated equipment and computational methods, expansion microscopy (ExM) is unique in that it provides increase in resolution by physically expanding the sample embedded in a water-swellable hydrogel. ExM has rapidly grown in prevalence, owing to its ease of use and economic nature – all necessary reagents are commercially available, and samples may be imaged in large volume on conventional fluorescence microscopes. Here, we demonstrate the power of expansion microscopy on imaging synaptic connections onto a dopaminergic neuron, in the mouse substantia nigra pars compacta, with nanoscale resolution.

Cocaine Selectively Reorganizes Excitatory Inputs to Substantia Nigra Pars Compacta Dopamine Neurons.

Substantia nigra pars compacta (SNc) dopamine neurons and their targets are involved in addiction and cue-induced relapse. However, afferents onto SNc dopamine neurons themselves appear insensitive to drugs of abuse, such as cocaine, when afferents are collectively stimulated electrically. This contrasts with ventral tegmental area (VTA) dopamine neurons, whose glutamate afferents react robustly to cocaine. We used an optogenetic strategy to isolate identified SNc inputs and determine whether cocaine sensitivity in the mouse SNc circuit is conferred at the level of three glutamate afferents: dorsal raphé nucleus (DR), pedunculopontine nucleus (PPN), and subthalamic nucleus (STN). We found that excitatory afferents to SNc dopamine neurons are sensitive to cocaine in an afferent-specific manner. A single exposure to cocaine in vivo led to PPN-innervated synapses reducing the AMPA-to-NMDA receptor-mediated current ratio. In contrast to work in the VTA, this was due to increased NMDA receptor function with no change in AMPA receptor function. STN synapses showed a decrease in calcium-permeable AMPA receptors after cocaine, but no change in the AMPA-to-NMDA ratio. Cocaine also increased the release probability at DR-innervated and STN-innervated synapses, quantified by decreases in paired-pulse ratios. However, release probability at PPN-innervated synapses remained unaffected. By examining identified inputs, our results demonstrate a functional distribution among excitatory SNc afferent nuclei in response to cocaine, and suggest a compelling architecture for differentiation and separate parsing of inputs within the nigrostriatal system.SIGNIFICANCE STATEMENT Prior studies have established that substantia nigra pars compacta (SNc) dopamine neurons are a key node in the circuitry that drives addiction and relapse, yet cocaine apparently has no effect on electrically stimulated excitatory inputs. Our study is the first to demonstrate the functional impact of a drug of abuse on synaptic mechanisms of identified afferents to the SNc. Optogenetic dissection of inputs originating from dorsal raphé, pedunculopontine, and subthalamic nuclei were tested for synaptic modifications following in vivo cocaine exposure. Our results demonstrate that cocaine differentially induces modifications to SNc synapses depending on input origin. This presents implications for understanding dopamine processing of motivated behavior; most critically, it indicates that dopamine neurons selectively modulate signal reception processed by afferent nuclei.

Transient Activation of GABAB Receptors Suppresses SK Channel Currents in Substantia Nigra Pars Compacta Dopaminergic Neurons.

Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) are richly innervated by GABAergic neurons. The postsynaptic effects of GABA on SNc DA neurons are mediated by a mixture of GABAA and GABAB receptors. Although activation of GABAA receptors inhibits spike generation, the consequences of GABAB receptor activation are less well characterized. To help fill this gap, perforated patch recordings were made from young adult mouse SNc DA neurons. Sustained stimulation of GABAB receptors hyperpolarized SNc DA neurons, as previously described. However, transient stimulation of GABAB receptors by optical uncaging of GABA did not; rather, it reduced the opening of small-conductance, calcium-activated K+ (SK) channels and increased the irregularity of spiking. This modulation was attributable to inhibition of adenylyl cyclase and protein kinase A. Thus, because suppression of SK channel activity increases the probability of burst spiking, transient co-activation of GABAA and GABAB receptors could promote a pause-burst pattern of spiking.

Environment- and activity-dependent dopamine neurotransmitter plasticity in the adult substantia nigra

The ability of neurons to change the amount or type of neurotransmitter they use, or ‘neurotransmitter plasticity’, is an emerging new form of adult brain plasticity. For example, it has recently been shown that neurons in the adult rat hypothalamus up- or down-regulate dopamine (DA) neurotransmission in response to the amount of light the animal receives (photoperiod), and that this in turn affects anxiety- and depressive-like behaviors (Dulcis et al., 2013). In this Chapter I consolidate recent evidence from my laboratory suggesting neurons in the adult mouse substantia nigra pars compacta (SNc) also undergo DA neurotransmitter plasticity in response to persistent changes in their electrical activity, including that driven by the mouse's environment or behavior. Specifically, we have shown that the amounts of tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) gene promoter activity, TH mRNA and TH protein in SNc neurons increases or decreases after ∼20h of altered electrical activity. Also, infusion of ion-channel agonists or antagonists into the midbrain for 2 weeks results in ∼10% (∼500 neurons) more or fewer TH immunoreactive (TH+) SNc neurons, with no change in the total number of SNc neurons (TH+ and TH−). Targeting ion-channels mediating cell-autonomous pacemaker activity in, or synaptic input and afferent pathways to, SNc neurons are equally effective in this regard. In addition, exposing mice to different environments (sex pairing or environment enrichment) for 1–2 weeks induces ∼10% more or fewer TH+ SNc (and ventral tegmental area or VTA) neurons and this is abolished by concurrent blockade of synaptic transmission in midbrain. Although further research is required to establish SNc (and VTA) DA neurotransmitter plasticity, and to determine whether it alters brain function and behavior, it is an exciting prospect because: (1) It may play important roles in movement, motor learning, reward, motivation, memory and cognition; and (2) Imbalances in midbrain DA cause symptoms associated with several prominent brain and behavioral disorders such as schizophrenia, addiction, obsessive-compulsive disorder, depression, Parkinson’s disease and attention-deficit and hyperactivity disorder. Midbrain DA neurotransmitter plasticity may therefore play a role in the etiology of these symptoms, and might also offer new treatment options.

Inhibition of A-type potassium current by the peptide toxin SNX-482.

SNX-482, a peptide toxin isolated from tarantula venom, has become widely used as an inhibitor of Cav2.3 voltage-gated calcium channels. Unexpectedly, we found that SNX-482 dramatically reduced the A-type potassium current in acutely dissociated dopamine neurons from mouse substantia nigra pars compacta. The inhibition persisted when calcium was replaced by cobalt, showing that it was not secondary to a reduction of calcium influx. Currents from cloned Kv4.3 channels expressed in HEK-293 cells were inhibited by SNX-482 with an IC50 of <3 nM, revealing substantially greater potency than for SNX-482 inhibition of Cav2.3 channels (IC50 20-60 nM). At sub-saturating concentrations, SNX-482 produced a depolarizing shift in the voltage dependence of activation of Kv4.3 channels and slowed activation kinetics. Similar effects were seen on gating of cloned Kv4.2 channels, but the inhibition was less pronounced and required higher toxin concentrations. These results reveal SNX-482 as the most potent inhibitor of Kv4.3 channels yet identified. Because of the effects on both Kv4.3 and Kv4.2 channels, caution is needed when interpreting the effects of SNX-482 on cells and circuits where these channels are present.

Ghrelin inhibits LPS-induced release of IL-6 from mouse dopaminergic neurones

BackgroundGhrelin is an orexigenic stomach hormone that acts centrally to increase mid-brain dopamine neurone activity, amplify dopamine signaling and protect against neurotoxin-induced dopamine cell death in the mouse substantia nigra pars compacta (SNpc). In addition, ghrelin inhibits the lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from peripheral macrophages, T-cells and from LPS stimulated microglia. Here we sought to determine whether ghrelin attenuates pro-inflammatory cytokine release from dopaminergic neurones.FindingsThe dopaminergic SN4741 cell-line, which derives from the mouse substantia nigra (SN) and expresses the ghrelin-receptor (growth hormone secretagogue receptor (GHS-R)) and the ghrelin-O-acyl transferase (GOAT) enzyme, was used to determine the neuro-immunomodulatory action of ghrelin. We induced innate immune activation via LPS challenge (1 μg/ml) of SN4741 neurones that had been pre-cultured in the presence or absence of ghrelin (1, 10, 100 nM) for 4 h. After 24 h supernatants were collected for detection of IL-1 beta (IL-1β ), TNF alpha (TNF-α) and IL-6 cytokines via enzyme linked immunosorbent assay (ELISA) analysis. Nuclear translocation of the transcription factor nuclear factor kappa B (NF-κB) was analyzed by Western blotting, and to determine viability of treatments a cell viability assay and caspase-3 immunohistochemistry were performed.We provide evidence that while IL-1β and TNF-α were not detectable under any conditions, SN4741 neurones constitutively released IL-6 under basal conditions and treatment with LPS significantly increased IL-6 secretion. Pre-treatment of neurones with ghrelin attenuated LPS-mediated IL-6 release at 24 h, an affect that was inhibited by the GHS-R antagonist [D-Lys3]-GHRP-6. However, while ghrelin pre-treatment attenuated the LPS-mediated increase in NF-κB, there was no alteration in its nuclear translocation. Cell viability assay and caspase-3 immunocytochemistry demonstrated that the results were independent from activation of cytotoxic and/or apoptotic mechanisms in the neuronal population, respectively.ConclusionOur results provide evidence that the gut-hormone, ghrelin, attenuates IL-6 secretion to LPS challenge in mid-brain dopaminergic neurones. These data suggest that ghrelin may protect against dopaminergic SN nerve cell damage or death via modulation of the innate immune response.

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1 μM tetrodotoxin decreased TH expression beginning around 20 h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300 nM apamin increased TH mRNA and protein between 12 and 24 h later in slices of adult midbrain. Two-week infusions of 300 nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ∼20 h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.

Blockade of PrRP attenuates MPTP-induced toxicity in mice

Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of the orphan G-protein coupled receptor hGR3. PrRP has been shown to be involved in the regulation of food intake, stress responses, prolactin secretion and release, blood pressure, and the opioid system. Here we report that PrRP and its receptor, GPR10, were found in the mouse substantia nigra pars compacta (SNpc), the main location of dopaminergic (DA) neurons of the nigrostriatal system. We generated PrRP knockout (KO) mice, and then treated PrRP KO mice and their wild type (WT) littermates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neuron toxin that selectively damages DA neurons in the SNpc. We found that PrRP KO mice were resistant to MPTP-induced lesions of the nigrostriatal system. These effects were further confirmed by the intracerebroventricular injection of P2L-1C, a monoclonal antibody against PrRP into mice. Taken together, our data established a critical role of PrRP in MPTP intoxication in mice.

P53 Mediates Nontranscriptional Cell Death in Dopaminergic Cells in Response to Proteasome Inhibition

Proteasome dysfunction has been demonstrated in Parkinson disease (PD), and proteasome inhibitors have been shown to induce degeneration of dopaminergic neurons in vitro and in vivo. The mechanism whereby proteasome dysfunction leads to dopaminergic cell death, however, is unknown. In this study, we show that proteasome inhibition in both PC12 cells and dopaminergic neurons derived from embryonic stem cells is associated with mitochondrial membrane permeabilization, activation of caspase-3, and nuclear changes consistent with apoptosis. Prior to the emergence of apoptotic features, we found that proteasome inhibition induced increased levels of phosphorylated p53. Inhibition of p53 by pifithrin-alpha or by RNA interference prevented mitochondrial membrane permeabilization and cytotoxicity. There was no increase in p53 mRNA in proteasome-inhibited cells, suggesting that p53 was increased in a transcription-independent manner. Further, there was no increase in Puma or Bax mRNA and p53 co-immunoprecipitated with Bcl-xL and Mdm2. These findings suggest that p53 mediates cell death by way of a direct mitochondrial effect in this model. We also observed increased levels of phosphorylated p53 in dopamine neurons of the substantia nigra pars compacta of mice following systemic administration of a proteasome inhibitor. These changes preceded degeneration of dopaminergic neurons. Increased phosphorylated p53 was also demonstrated in the substantia nigra pars compacta of post-mortem PD brains. These results suggest that abnormalities in p53 signaling play a role in dopaminergic cell death induced by proteasome inhibition and may be relevant to neurodegeneration in PD.

Evidence of Apoptosis in the Subventricular Zone and Rostral Migratory Stream in the MPTP Mouse Model of Parkinson Disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to create animal models of Parkinson disease. There is conflicting evidence on the occurrence of apoptosis induced by MPTP in the mouse substantia nigra pars compacta. We demonstrated that a single acute injection of MPTP induced apoptosis in the subventricular zone (SVZ) and rostral migratory stream (RMS) in the adult C57BL/6 mouse brain. The number of TUNEL-positive cells peaked at 24 hours after injection and decreased thereafter, paralleling the change in the number of cleaved caspase-3-positive cells after MPTP injection. Results of immunohistochemistry and ultrastructural analyses indicated that the majority of apoptotic cells in the SVZ and RMS were migrating neuroblasts (type A cells), whereas a few were astrocytes (type B cells). No apoptosis occurred in transit-amplifying progenitors (type C cells). The decrease in A cell numbers was most marked on day 2 and lasted to day 8 after the administration. A rapid and transient phagocytosis of apoptotic cells by microglial cells was demonstrated to parallel the MPTP-induced apoptosis. The present findings provide new insight into the extensive neurotoxicity of MPTP and may be valuable in reevaluating the MPTP mouse model of Parkinson disease.

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.

Calcium mobilization elicited by two types of nicotinic acetylcholine receptors in mouse substantia nigra pars compacta

Nicotinic acetylcholine receptors (nAChRs) are expressed in the midbrain ascending dopaminergic system, a target of many addictive drugs. Here we assessed the intracellular Ca2+ level by imaging fura-2-loaded cells in substantia nigra pars compacta in mouse brain slices, and we examined the influence on this level of prolonged exposures to nicotine using mice lacking the nAChR beta2-subunit. In control cells, superfusion with nicotine (10-100 microM) caused a long-lasting rise of intracellular Ca2+ level which depended on extracellular Ca2+. This nicotinic response was almost completely absent in beta2-/- mutant mice, leaving a small residual response to a high concentration (100 microM) of nicotine which was inhibited by the alpha7-subunit-selective antagonist, methyllycaconitine. Conversely, the alpha7-subunit-selective agonist choline (10 mM) caused a methyllycaconitine-sensitive increase in intracellular Ca2+ level both in wild-type and beta2-/- mutant mice. Nicotine-elicited Ca2+ mobilization was reduced by the Na+ channel blocker tetrodotoxin (TTX) and by T-type Ca2+ channel blocking agents, whereas the choline-elicited Ca2+ increase was insensitive to TTX. Neither nicotine nor choline produced Ca2+ increase following inhibition of the release of Ca2+ from intracellular stores by dantrolene. These results demonstrate that in nigral dopaminergic neurons, nicotine can elicit Ca2+ mobilization via activation of two distinct nAChR subtypes: that of beta2-subunit-containing nAChR followed by activation of Na+ channel and T-type Ca2+ channels, and/or activation of alpha7-subunit-containing nAChR. The Ca2+ influx due to nAChR activation is subsequently amplified by the recruitment of intracellular Ca2+ stores. This Ca2+ mobilization may possibly contribute to the long-term effects of nicotine on the dopaminergic system.