Biological Mechanism-based Neurology and Psychiatry: A BACE1/2 and Downstream Pathway Model.

In oncology, comprehensive omics and functional enrichment studies have led to an extensive profiling of (epi)genetic and neurobiological alterations that can be mapped onto a single tumor's clinical phenotype and divergent clinical phenotypes expressing common pathophysiological pathways. Consequently, molecular pathway-based therapeutic interventions for different cancer typologies, namely tumor type- and site-agnostic treatments, have been developed, encouraging the real-world implementation of a paradigm shift in medicine. Given the breakthrough nature of the new-generation translational research and drug development in oncology, there is an increasing rationale to transfertilize this blueprint to other medical fields, including psychiatry and neurology. In order to illustrate the emerging paradigm shift in neuroscience, we provide a state-of-the-art review of translational studies on the ß-site amyloid precursor protein cleaving enzyme (BACE) and its most studied downstream effector, neuregulin, which are molecular orchestrators of distinct biological pathways involved in several neurological and psychiatric diseases. This body of data aligns with the evidence of a shared genetic/biological architecture among Alzheimer's disease, schizoaffective disorder, and autism spectrum disorders. To facilitate a forward-looking discussion about a potential first step towards the adoption of biological pathway-based, clinical symptom-agnostic, categorization models in clinical neurology and psychiatry for precision medicine solutions, we engage in a speculative intellectual exercise gravitating around BACE-related science, which is used as a paradigmatic case here. We draw a perspective whereby pathway-based therapeutic strategies could be catalyzed by highthroughput techniques embedded in systems-scaled biology, neuroscience, and pharmacology approaches that will help overcome the constraints of traditional descriptive clinical symptom and syndrome-focused constructs in neurology and psychiatry.

A2A Receptor Dysregulation in Dystonia DYT1 Knock-Out Mice.

We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/- knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3-0.4 µm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/-, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/-, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/-, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/-, opposite changes of A2A receptors' expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia.

Insights on the Functional Interaction between Group 1 Metabotropic Glutamate Receptors (mGluRI) and ErbB Receptors.

It is well-appreciated that phosphorylation is an essential post-translational mechanism of regulation for several proteins, including group 1 metabotropic glutamate receptors (mGluRI), mGluR1, and mGluR5 subtypes. While contributions of various serine/threonine protein kinases on mGluRI modulation have been recognized, the functional role of tyrosine kinases (TKs) is less acknowledged. Here, while describing current evidence supporting that mGluRI are targets of TKs, we mainly focus on the modulatory roles of the ErbB tyrosine kinases receptors-activated by the neurotrophic factors neuregulins (NRGs)-on mGluRI function. Available evidence suggests that mGluRI activity is tightly dependent on ErbB signaling, and that ErbB's modulation profoundly influences mGluRI-dependent effects on neurotransmission, neuronal excitability, synaptic plasticity, and learning and memory processes.

Systemic activation of Nrf2 pathway in Parkinson's disease.

BACKGROUND: Preclinical studies underlined the relevance of Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor pathway in the pathogenesis of Parkinson's disease (PD). OBJECTIVE: The objective of this study was to explore Nrf2 pathway in vivo in PD, looking for novel disease biomarkers and therapeutic targets. METHODS: The levels of Nrf2, the downstream effectors (NAD(P)H dehydrogenase [quinone] 1 (Nqo1) enzyme, glutathione metabolism enzymes Glutamate-cysteine ligase (GCL) and Glutathione Reductase (GR)), the upstream activators (redox state and mitochondrial dysfunction), and α-synuclein oligomers were assessed in the blood leukocytes of PD patients comparatively to controls. Biochemical data were correlated to clinical parameters. RESULTS: In PD, Nrf2 was highly transcribed and expressed as well as its target effectors. The mitochondrial complex I activity was reduced and the oxidized form of glutathione prevailed, disclosing the presence of pathway's activators. Also, α-synuclein oligomers levels were increased. Nrf2 transcript and oligomers levels correlated with PD duration. CONCLUSIONS: Blood leukocytes mirror pathogenic mechanisms of PD, showing the systemic activation of the Nrf2 pathway and its link with synucleinopathy and clinical events. © 2019 International Parkinson and Movement Disorder Society.

Hippocampal epileptogenesis in autoimmune encephalitis.

OBJECTIVE: Autoantibody-mediated forms of encephalitis (AE) include neurological disorders characterized by subacute memory loss, movement disorders, and, often, frequent, focal epileptic seizures. Yet, the electrophysiological effects of these autoantibodies on neuronal function have received little attention. In this study, we assessed the effects of CSF containing autoantibodies on intrinsic and extrinsic properties of hippocampal neurons, to define their epileptogenic potential. METHODS: We compared the effects of CSF containing leucine-rich glioma inactivated 1 (LGI1), contactin-associated protein-like 2 (CASPR2), and γ-aminobutyric acid receptor B (GABAB R) antibodies on ex vivo electrophysiological parameters after stereotactic hippocampal inoculation into mice. Whole-cell patch-clamp and extracellular recordings from CA1 pyramidal neurons and CA3-CA1 field recordings in ex vivo murine brain slices were used to study neuronal function. RESULTS: By comparison to control CSF, AE CSFs increased the probability of glutamate release from CA3 neurons. In addition, LGI1- and CASPR2 antibodies containing CSFs induced epileptiform activity at a population level following Schaffer collateral stimulation. CASPR2 antibody containing CSF was also associated with higher spontaneous firing of CA1 pyramidal neurons. On the contrary, GABAB R antibody containing CSF did not elicit changes in intrinsic neuronal activity and field potentials. INTERPRETATION: Using patient CSF, we have demonstrated that the AE-associated antibodies against LGI1 and CASPR2 are able to increase hippocampal CA1 neuron excitability, facilitating epileptiform activity. These findings provide in vivo pathogenic insights into neuronal dysfunction in these conditions.

Impact of Pharmacological Inhibition of Hydrogen Sulphide Production in the SOD1G93A-ALS Mouse Model.

A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)-a systemic dual inhibitor of cystathionine-ß-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.

L-DOPA-quinone Mediated Recovery from GIRK Channel Firing Inhibition in Dopaminergic Neurons.

The oxidative degeneration of dopamine-releasing (DAergic) neurons in the substantia nigra pars compacta (SNc) has attracted much interest in preclinical research, due to its involvement in Parkinson's disease manifestations. Evidence exists on the participation of quinone derivatives in mitochondrial dysfunction, alpha synuclein protein aggregation, and protein degradation. With the aim to investigate the role of L-DOPA-quinone in DAergic neuron functions, we synthesized L-DOPA-quinone by use of 2-iodoxybenzoic acid and measured its activity in recovery from dopamine-mediated firing inhibition of SNc neurons. Noteworthy, L-DOPA-quinone counteracts firing inhibition in SNc DAergic neurons caused by GIRK opening. A possible mechanism to explain the effect of L-DOPA-quinone on GIRK channel has been proposed by computational models. Overall, the study showed the possibility that L-DOPA-quinone stabilizes GIRK in a preopen conformation through formation of a covalent adduct with cysteine-65 on the GIRK2 subunit of the protein.

Thymomatous myasthenia gravis: novel association with HLA DQB1*05:01 and strengthened evidence of high clinical and serological severity.

BACKGROUND: The relative prevalence of myasthenia gravis (MG) subtypes is changing, and their differential features and association with HLA class II alleles are not completely understood. METHODS: Age at onset, presence/absence of autoantibodies (Ab) and thymoma were retrospectively considered in 230 adult Italian patients. Clinical severity, assessed by MGFA scale, and the highest Ab titer were recorded. Furthermore, we performed low/high resolution typing of HLA-DRB1 and HLA-DQB1 alleles to detect associations of these loci with MG subtypes. RESULTS: There were two peaks of incidence: under 41 years of age, with female preponderance, and over 60 years, with higher male prevalence. The former group decreased and the latter increased significantly when comparing onset period 2008-2015 to 2000-2007. Thymomatous (TMG) patients showed a higher prevalence of severe phenotype and significantly higher anti-AChR Ab titer than non-thymomatous (NTMG) patients. Among the latter, those with onset after 60 years of age (LO-NTMG) displayed significantly higher Ab titers but lower MGFA grade compared to early-onset patients (< 41 years; EO-NTMG). Significant associations were found between HLA DQB1*05:01 and TMG patients and between DQB1*05:02 and DRB1*16 alleles and LO-NTMG with anti-AChR Ab. CONCLUSIONS: Two distinct cutoffs (< 41 and > 60 years) conveniently define EO-NTMG and LO-NTMG, with different characteristics. LO-NTMG is the most frequent disease subtype, with an increasing incidence. TMG patients reach higher clinical severity and higher antibody titers than NTMG patients. Moreover, TMG and LO-NTMG with anti-AChR Ab differ in their HLA-DQ association, providing further evidence that these two forms may have different etiologic mechanisms.

Treating TB human neuroectodermal cell line with retinoic acid induces the appearance of neuron-like voltage-gated ionic currents.

TB is a cell line derived from the cerebrospinal fluid sample of a patient with primary leptomeningeal melanomatosis. Our previous immunological and ultrastructural analysis revealed that TB cells differentiate towards a neuronal phenotype when grown in vitro up to 7 days in presence of 10 µM all-trans retinoic acid (RA). Recently, we reported that TB cells are sensitive to the cytotoxic effects of ß-amyloid peptides, activating the cytosolic phospholipase A2. To date, it is not known if RA, in addition to inducing morphological changes, also causes functional modification in TB cells, by regulating voltage-gated ionic currents. To this purpose, we performed electrophysiological characterization of undifferentiated (TB) and differentiated (RA-TB) cells by means of whole-cell patch clamp recordings. Upon depolarizing stimuli, both groups displayed voltage-gated K+ outward currents of similar amplitude. By contrast, the low amplitude voltage-gated Na+ currents recorded in undifferentiated TB cells were largely up-regulated by RA exposure. This current was strongly reduced by TTX and lidocaine and completely abolished by removal of extracellular sodium. Furthermore, treatment with RA caused the appearance of a late-onset inward current carried by Ca2+ ions in a subpopulation of TB cells. This current was not affected by removal of extracellular Na+ and was completely blocked by Cd2+, a broad-spectrum blocker of Ca2+ currents. Altogether, our results indicate that RA-differentiation of TB cells induces functional changes by augmenting the amplitude of voltage-gated sodium current and by inducing, in a subpopulation of treated cells, the appearance of a voltage-gated calcium current.

On the Modulatory Roles of Neuregulins/ErbB Signaling on Synaptic Plasticity.

Neuregulins (NRGs) are a family of epidermal growth factor-related proteins, acting on tyrosine kinase receptors of the ErbB family. NRGs play an essential role in the development of the nervous system, since they orchestrate vital functions such as cell differentiation, axonal growth, myelination, and synapse formation. They are also crucially involved in the functioning of adult brain, by directly modulating neuronal excitability, neurotransmission, and synaptic plasticity. Here, we provide a review of the literature documenting the roles of NRGs/ErbB signaling in the modulation of synaptic plasticity, focusing on evidence reported in the hippocampus and midbrain dopamine (DA) nuclei. The emerging picture shows multifaceted roles of NRGs/ErbB receptors, which critically modulate different forms of synaptic plasticity (LTP, LTD, and depotentiation) affecting glutamatergic, GABAergic, and DAergic synapses, by various mechanisms. Further, we discuss the relevance of NRGs/ErbB-dependent synaptic plasticity in the control of brain processes, like learning and memory and the known involvement of NRGs/ErbB signaling in the modulation of synaptic plasticity in brain's pathological conditions. Current evidence points to a central role of NRGs/ErbB receptors in controlling glutamatergic LTP/LTD and GABAergic LTD at hippocampal CA3-CA1 synapses, as well as glutamatergic LTD in midbrain DA neurons, thus supporting that NRGs/ErbB signaling is essential for proper brain functions, cognitive processes, and complex behaviors. This suggests that dysregulated NRGs/ErbB-dependent synaptic plasticity might contribute to mechanisms underlying different neurological and psychiatric disorders.

Epilepsy, amyloid-ß, and D1 dopamine receptors: a possible pathogenetic link?

Experimental and clinical observations indicate that amyloid-ß1-42 (Aß1-42) peptide not only represents a major actor in neurodegenerative mechanisms but also induce hyperexcitation in individual neurons and neural circuits. In this abnormal excitability, possibly leading to seizures, the D1 dopamine (DA) receptors may play a role. Cerebrospinal fluid levels of Aß1-42 were measured in patients with late-onset epilepsy of unknown etiology. Moreover, the effect of amyloid peptide on the hippocampal epileptic threshold and synaptic plasticity and its link to D1 receptor function were tested in experimental mouse model of cerebral amyloidosis and in acute model of Aß1-42-induced neurotoxicity. Among 272 evaluated epileptic patients, aged >55 years, 35 suffered from late-onset epilepsy of unknown etiology. In these subjects, cerebrospinal fluid Aß1-42 levels were measured. The effects of Aß1-42, amyloid oligomers, and D1 receptor modulation on epileptic threshold were analyzed by electrophysiological recordings in the dentate gyrus of mice hippocampal slices. We found that Aß1-42 levels were significantly decreased in cerebrospinal fluid of patients with late-onset epilepsy of unknown etiology with respect to controls suggesting the cerebral deposition of this peptide in these patients. Aß1-42 enhanced epileptic activity in mice through a mechanism involving increased surface expression of D1 receptor, and this effect was mimicked by D1 receptor stimulation and blocked by SCH 23390, a D1 receptor antagonist. Aß1-42 may contribute to the pathophysiology of late-onset epilepsy of unknown origin. Our preclinical findings indicate that the D1 receptor is involved in mediating the epileptic effects of Aß1-42. This novel link between Aß1-42 and D1 receptor signaling might represent a potential therapeutic target.

Evidence of hydrogen sulfide involvement in amyotrophic lateral sclerosis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease whose pathophysiological deficits, causing impairment in motor function, are largely unknown. Here we propose that hydrogen sulfide (H2 S), as a glial-released inflammatory factor, contributes to ALS-mediated motor neuron death. METHODS: H2 S concentrations were analyzed in the cerebrospinal fluid of 37 sporadic ALS patients and 14 age- and gender-matched controls, in tissues of a familial ALS (fALS) mouse model, and in spinal cord culture media by means of a specific and innovative high-performance liquid chromatography method. The effects of H2 S on motor neurons cultures was analyzed immunohistochemically and by patch clamp recordings and microfluorometry. RESULTS: We found a significantly high level of H2 S in the spinal fluid of the ALS patients. Consistently, we found increased levels of H2 S in the tissues and in the media from mice spinal cord cultures bearing the fALS mutation SOD1G93A. In addition, NaHS, an H2 S donor, added to spinal culture, obtained from control C57BL/6J mice, is toxic for motor neurons, and induces an intracellular Ca(2+) increase, attenuated by the intracytoplasmatic application of adenosine triphosphate. We further show that H2 S is mainly released by astrocytes and microglia. INTERPRETATION: This study unravels H2 S as an astroglial mediator of motor neuron damage possibly involved in the cellular death characterizing ALS.

Insulin receptor ß-subunit haploinsufficiency impairs hippocampal late-phase LTP and recognition memory.

The insulin receptor (IR) is a protein tyrosine kinase playing a pivotal role in the regulation of peripheral glucose metabolism and energy homoeostasis. IRs are also abundantly distributed in the cerebral cortex and hippocampus, where they regulate synaptic activity required for learning and memory. As the major anabolic hormone in mammals, insulin stimulates protein synthesis partially through the activation of the PI3K/Akt/mTOR pathway, playing fundamental roles in neuronal development, synaptic plasticity and memory. Here, by means of a multidisciplinary approach, we report that long-term synaptic plasticity and recognition memory are impaired in IR ß-subunit heterozygous mice. Since IR expression is diminished in type-2 diabetes as well as in Alzheimer's disease (AD) patients, these data may provide a mechanistic link between insulin resistance, impaired synaptic transmission and cognitive decline in humans with metabolic disorders.

Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D(1)R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.

Dopamine D2 receptor dysfunction is rescued by adenosine A2A receptor antagonism in a model of DYT1 dystonia.

DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins. Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice. Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.

Acute action of rotenone on nigral dopaminergic neurons--involvement of reactive oxygen species and disruption of Ca2+ homeostasis.

Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 microm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K(+) channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).

Memantine inhibits ATP-dependent K+ conductances in dopamine neurons of the rat substantia nigra pars compacta.

1-Amino-3,5-dimethyl-adamantane (memantine) is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist used in clinical practice to treat neurodegenerative disorders that could be associated with excitotoxic cell death. Because memantine reduces the loss of dopamine neurons of the substantia nigra pars compacta (SNc) in animal models of Parkinson's disease, we examined the effects of this drug on dopamine cells of the SNc. Besides inhibition of NMDA receptor-mediated currents, memantine (30 and 100 microM) increased the spontaneous firing rate of whole-cell recorded dopamine neurons in a midbrain slice preparation. Occasionally, a bursting activity was observed. These effects were independent from the block of NMDA receptors and were prevented in neurons dialyzed with a high concentration of ATP (10 mM). An increase in firing rate was also induced by the ATP-sensitive potassium (K(ATP)) channel antagonist tolbutamide (300 microM), and this increase occluded further effects of memantine. In addition, K(ATP) channel-mediated outward currents, induced by hypoxia, were inhibited by memantine (30 and 100 microM) in the presence of the NMDA receptor antagonist (5S, 10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 microM). An increase in the spontaneous firing rate by memantine was observed in dopamine neurons recorded with extracellular planar 8 x 8 multielectrodes in conditions of hypoglycemia. These results highlight K(ATP) channels as possible relevant targets of memantine effects in the brain. Moreover, in view of a proposed role of K(ATP) conductances in dopamine neuron degeneration, they suggest another mechanism of action underlying the protective role of memantine in Parkinson's disease.

Trace amines depress GABA B response in dopaminergic neurons by inhibiting G-betagamma-gated inwardly rectifying potassium channels.

Trace amines (TAs) are present in the central nervous system in which they up-regulate catecholamine release and are implicated in the pathogenesis of addiction, attention-deficit/hyper-activity disorder, Parkinson's disease, and schizophrenia. By using intracellular and patch-clamp recordings from dopaminergic cells in the rat midbrain slices, we report a depressant postsynaptic action of two TAs, beta-phenylethylamine (beta-PEA) and tyramine (TYR) on the GABA(B)-mediated slow inhibitory postsynaptic potential and baclofen-activated outward currents. beta-PEA and TYR activated G-proteins, interfering with the coupling between GABA(B) receptors and G-betagamma-gated inwardly rectifying potassium channels. This is the first demonstration that beta-PEA and TYR depress inhibitory synaptic potentials in neurons of the central nervous system, supporting their emerging role as neuromodulators.

Methamidophos transiently inhibits neuronal nicotinic receptors of rat substantia nigra dopaminergic neurons via open channel block.

The use of acetylcholinesterase (AChE) inhibitors is the primary therapeutic strategy in the treatment of Alzheimer's disease. However, these drugs have been reported to have effects beyond the simple stimulation of neuronal acetylcholine receptors (AChRs) by elevated acetylcholine (ACh), interfering directly with the nAChR. Therefore, a pure pharmacological blockade of AChE is not usually obtained. In this study, the patch-clamp technique was utilized to determine the effects of methamidophos, a pesticide that is considered a selective AChE inhibitor, on nAChRs of substantia nigra dopaminergic neurons. In spite of the fact that methamidophos has been reported to be devoid of direct nicotinic actions, our main observation was that it selectively and reversibly blocked nAChR responses, without directly affecting the holding current. Methamidophos produced a downward shift in the dose response curve for nicotine; the mechanism accounting for this non-competitive antagonism was open channel block, in view of its voltage dependence. Pre-treatment with vesamicol did not prevent the reduction of nicotine-induced currents, indicating that the effect on nAChRs was independent from the activity of methamidophos as a cholinesterase inhibitor. Our results conclude that methamidophos has a complex blocking action on neuronal nAChRs that is unlinked to the inhibition of AChE. Therefore, it should not be considered a selective AChE inhibitor and part of its toxic effects could reside in an interference with the nicotinic neurotransmission.

Donepezil modulates nicotinic receptors of substantia nigra dopaminergic neurones.

1. The effects of donepezil, one of the most common cholinesterase inhibitors used for treatment of Alzheimer's disease, were studied on nicotinic receptors (nAChRs)-mediated postsynaptic currents, in dopaminergic neurons of the substantia nigra pars compacta, using the patch-clamp recording technique in slice preparations. 2. Donepezil (10-100 microM) selectively and reversibly depressed nicotine currents, induced by brief puffer pulses, through a glass micropipette positioned above the slice. 3. The peak amplitude fading of the responses generated by repeated test applications of low doses of nicotine was accelerated by donepezil, while it slowed the recovery of nicotine currents after a large, desensitising, dose of the same agonist. 4. Donepezil depressed even maximal responses to nicotine, revealing a noncompetitive mechanism of action; moreover, the inhibition of nAChRs was voltage and time independent. 5. Pretreatment with vesamicol or methamidophos did not prevent the reduction of nicotine-induced currents. The data indicated direct effect on nAChR, independent from the activity of donepezil as cholinesterase inhibitor.