No abstract

The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch‐clamp recording. Pharmacologically isolated GABAA receptor‐mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner (IC50, 10.0 μm). A D2‐like receptor agonist, quinpirole (3–30 μm), also suppressed the IPSCs, whereas a D1‐like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D2‐like receptor antagonist, blocked the DA‐induced suppression of IPSCs (apparent dissociation constant (KB), 0.36 μm), while a D1‐like receptor antagonist, SCH 23390 (10 μm), had no effect. DA (30 μm) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABAA receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca2+ was replaced by Mg2+ (5 mm), indicating that DA affected the Ca2+ entry into the presynaptic terminal. An N‐type Ca2+ channel selective blocker, ω‐conotoxin GVIA (ω‐CgTX, 3 μm), suppressed IPSCs by 65.4%, whereas a P/Q‐type Ca2+ channel selective blocker, ω‐agatoxin IVA (ω‐Aga‐IVA, 200 nm), suppressed IPSCs by 78.4%. Simultaneous application of both blockers suppressed IPSCs by 95.9%. Assuming a 3rd power relationship between Ca2+ concentration and transmitter release, the contribution of N‐, P/Q‐ and other types of Ca2+ channels to presynaptic Ca2+ entry is estimated to be, respectively, 29.8, 40.0 and 34.5% at this synapse. After the application of ω‐CgTX, DA (30 μm) no longer affected IPSCs. In contrast, ω‐Aga‐IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N‐type Ca2+ channels. A G protein alkylating agent, N‐ethylmaleimide (NEM), significantly reduced the DA‐induced suppression of IPSCs. These results suggest that presynaptic D2‐like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down‐regulate GABA release by selectively blocking N‐type Ca2+ channels through NEM‐sensitive G proteins.

Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12–/–) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12–/– bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12–/– mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P(2) phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a(-/-) mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P(2), PtdIns(3,4,5)P(3) and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P(2) metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P(2) metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.

The effects of dopamine (DA) on non‐NMDA glutamatergic transmission onto dopaminergic neurones in the ventral tegmental area (VTA) were examined in rat midbrain slices using the whole‐cell patch‐clamp technique. EPSCs in dopaminergic neurones evoked by focal stimulation within the VTA were reversibly blocked by 5 μM CNQX in the presence of bicuculline (20 μM), strychnine (0.5 μM) and D‐amino‐5‐phosphonopentanoic acid (D‐AP5, 25 μM). Bath application of DA reduced the amplitude of EPSCs up to 65.1 ± 9.52% in a concentration‐dependent manner between 0.3‐1000 μM (IC50, 16.0 μM) without affecting the holding current at −60 mV measured using a Cs+‐filled electrode. The effect of DA on evoked EPSCs was mimicked by the D2‐like receptor agonist quinpirole but not by the D1‐like receptor agonist SKF 81297, and was antagonized by the D2‐like receptor antagonist sulpiride (KB, 0.96 μM), but not by the D1‐like receptor antagonist SCH 23390 (KB, 228.6 μM). Dopamine (30 μM) reduced the mean frequency of spontaneous miniature EPSCs (mEPSCs) without affecting their mean amplitude, and the DA‐induced effect on the mEPSCs was dependent on the external Ca2+ concentration. These results suggest that afferent glutamatergic fibres which terminate on VTA dopaminergic neurones possess presynaptic D2‐like receptors, activation of which inhibits glutamate release by reducing Ca2+ influx.

Gene abnormalities in RBFOX1, encoding an mRNA-splicing factor, have been shown to cause autism spectrum disorder and other neurodevelopmental disorders. Since pathophysiological significance of the dominant nuclear isoform in neurons, RBFOX1-isoform1 (iso1), remains to be elucidated, we performed comprehensive analyses of Rbfox1-iso1 during mouse corticogenesis. Knockdown of Rbfox1-iso1 by in utero electroporation caused abnormal neuronal positioning during corticogenesis, which was attributed to impaired migration. The defects were found to occur during radial migration and terminal translocation, perhaps due to impaired nucleokinesis. Axon extension and dendritic arborization were also suppressed in vivo in Rbfox1-iso1-deficient cortical neurons. In addition, electrophysiology experiments revealed significant defects in the membrane and synaptic properties of the deficient neurons. Aberrant morphology was further confirmed by in vitro analyses; Rbfox1-iso1-konckdown in hippocampal neurons resulted in the reduction of primary axon length, total length of dendrites, spine density and mature spine number. Taken together, this study shows that Rbfox1-iso1 plays an important role in neuronal migration and synapse network formation during corticogenesis. Defects in these critical processes may induce structural and functional defects in cortical neurons, and consequently contribute to the pathophysiology of neurodevelopmental disorders with RBFOX1 abnormalities.

Both D1R and D2R knock out (KO) mice of the major dopamine receptors show significant motor impairments. However, there are some discrepant reports, which may be due to the differences in genetic background and experimental procedures. In addition, only few studies directly compared the motor performance of D1R and D2R KO mice. In this paper, we examined the behavioral difference among N10 congenic D1R and D2R KO, and wild type (WT) mice. First, we examined spontaneous motor activity in the home cage environment for consecutive 5 days. Second, we examined motor performance using the rota-rod task, a standard motor task in rodents. Third, we examined motor ability with the Step-Wheel task in which mice were trained to run in a motor-driven turning wheel adjusting their steps on foothold pegs to drink water. The results showed clear differences among the mice of three genotypes in three different types of behavior. In monitoring spontaneous motor activities, D1R and D2R KO mice showed higher and lower 24 h activities, respectively, than WT mice. In the rota-rod tasks, at a low speed, D1R KO mice showed poor performance but later improved, whereas D2R KO mice showed a good performance at early days without further improvement. When first subjected to a high speed task, the D2R KO mice showed poorer rota-rod performance at a low speed than the D1R KO mice. In the Step-Wheel task, across daily sessions, D2R KO mice increased the duration that mice run sufficiently close to the spout to drink water, and decreased time to touch the floor due to missing the peg steps and number of times the wheel was stopped, which performance was much better than that of D1R KO mice. These incongruent results between the two tasks for D1R and D2R KO mice may be due to the differences in the motivation for the rota-rod and Step-Wheel tasks, aversion- and reward-driven, respectively. The Step-Wheel system may become a useful tool for assessing the motor ability of WT and mutant mice.

Basal forebrain (BF) nuclei, consisting of the vertical and horizontal limbs of diagonal band of Broca (HDBB), substantia innominata (SI) and nucleus of basalis (nB), form the principal source of cholinergic innervation to the cortical and subcortical brain regions (Rye et al. 1984). Pathologically, degeneration of these cholinergic neurones has been observed in patients with Alzheimer's disease (Coyle et al. 1983;Oyanagi et al. 1989). Morphological studies have shown that the BF region receives dopaminergic fibres from the ventral tegmental area, substantia nigra pars compacta and medial zona interna (Martinez-Murillo et al. 1988;Semba et al. 1988;Eaton et al. 1994). It has been electrophysiologically demonstrated that activation of presynaptic dopamine D 1 -like receptors reduces glutamate release onto magnocellular BF neurones, mainly cholinergic, in a calcium-dependent manner (Momiyama et al. 1996). Although multiple types of Ca 2+ channels including N-, P/Q-and R-type channels are involved in the fast synaptic transmission in the central nervous system (Luebke et al. 1993;Takahashi & Momiyama, 1993;Regehr & Mintz, 1994;Umemiya & Berger, 1994;Wheeler et al. 1994;Wu & Saggau, 1994;Wu et al. 1998), it remains to be elucidated whether activation of these D 1 -like receptors blocks specific subtypes of Ca 2+ channels. The aim of the present study is to identify which Ca 2+ -channel subtypes are involved in the D 1 -like receptor-mediated presynaptic inhibition of the non-NMDA glutamatergic transmission onto BF cholinergic neurones. In the present study, cholinergic BF neurones could be pre-labelled using Cy3-192IgG (Wu et al. 2000), a marker for p75 neurotrophic factor receptors (Hartig et al. 1998), as neurones