Identification of Optogenetically Activated Striatal Medium Spiny Neurons by Npas4 Expression

Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum.     

Selective Deletion of PTEN in Dopamine Neurons Leads to Trophic Effects and Adaptation of Striatal Medium Spiny Projecting Neurons

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson''s disease, and other neurodegenerative disorders.     

Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

Modulation of type 1 cannabinoid receptor (CB₁) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB₁ are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)) on arrestin2-, Gα_i/o-, Gβγ-, Gα_s-, and Gα_q-mediated intracellular signaling in the mouse STHdh^Q7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB₁. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα_i/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB₁ levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα_q-dependent pathways. CP55,940 and CBD both signaled through Gα_s. CP55,940, but not CBD, activated downstream Gα_s pathways via CB₁ targets. THC and CP55,940 promoted CB₁ internalization and decreased CB₁ protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB₁ leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.     

Segregation and Crosstalk of D1 Receptor-Mediated Activation of ERK in Striatal Medium Spiny Neurons upon Acute Administration of Psychostimulants

The convergence of corticostriatal glutamate and dopamine from the midbrain in the striatal medium spiny neurons (MSN) triggers synaptic plasticity that underlies reinforcement learning and pathological conditions such as psychostimulant addiction. The increase in striatal dopamine produced by the acute administration of psychostimulants has been found to activate not only effectors of the AC5/cAMP/PKA signaling cascade such as GluR1, but also effectors of the NMDAR/Ca²⁺/RAS cascade such as ERK. The dopamine-triggered effects on both these cascades are mediated by D1R coupled to Golf but while the phosphorylation of GluR1 is affected by reductions in the available amount of Golf but not of D1R, the activation of ERK follows the opposite pattern. This segregation is puzzling considering that D1R-induced Golf activation monotonically increases with DA and that there is crosstalk from the AC5/cAMP/PKA cascade to the NMDAR/Ca²⁺/RAS cascade via a STEP (a tyrosine phosphatase). In this work, we developed a signaling model which accounts for this segregation based on the assumption that a common pool of D1R and Golf is distributed in two D1R/Golf signaling compartments. This model integrates a relatively large amount of experimental data for neurons in vivo and in vitro. We used it to explore the crosstalk topologies under which the sensitivities of the AC5/cAMP/PKA signaling cascade to reductions in D1R or Golf are transferred or not to the activation of ERK. We found that the sequestration of STEP by its substrate ERK together with the insensitivity of STEP activity on targets upstream of ERK (i.e. Fyn and NR2B) to PKA phosphorylation are able to explain the experimentally observed segregation. This model provides a quantitative framework for simulation based experiments to study signaling required for long term potentiation in MSNs.     

Dopamine D2 Receptor Stimulation Potentiates PolyQ-Huntingtin-Induced Mouse Striatal Neuron Dysfunctions via Rho/ROCK-II Activation

Background

Huntington''s disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.

Methodology/Principal Findings

Using primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.

Conclusions/Significance

Our data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease.     

Synthesis and Release of Dopamine in Rat Striatal Slices: Requirement for Exogenous Tyrosine in the Medium

When incubated in a tyrosine-free medium, the tissue dopamine (DA) level of rat striatal slices increased by about 921 ± 15 pmol/mg protein during 90 min of preincubation. In contrast, the tissue-free tyrosine level declined only 130 pmol/mg protein in the same assay period. Depolarization of the slices with high K⁺ increased both DA and DOPAC outputs and depleted tissue DA level by about 75%. Although 60 min of resting after high K⁺ depolarization significantly restored the tissue DA levels, neither this restoration nor depolarization-induced DA release was altered by exogenous tyrosine. Similarly, failure of exogenous tyrosine was also observed during three successive depolarization periods of striatal slices. These results indicate that nigrostriatal dopaminergic neurons are able to synthesize and release the DA in the absence of exogenous tyrosine in the medium. Since the free tyrosine level in the slices does not seem to be a sufficient source, it is likely that tyrosine mobilized from its bound source(s) supports the DA synthesis under in vitro experimental conditions.     

Dopamine Reduction of GABA Currents in Striatal Medium-sized Spiny Neurons is Mediated Principally by the D<Subscript>1</Subscript> Receptor Subtype

Dopamine modulates voltage- and ligand-gated currents in striatal medium-sized neurons (MSNs) through the activation of D1- and D2-like family receptors. GABA_A receptor-mediated currents are reduced by D1 receptor agonists, but the relative contribution of D₁ or D₅ receptors in this attenuation has been elusive due to the lack of selective pharmacological agents. Here we examined GABA_A receptor-mediated currents and the effects of D1 agonists on MSNs from wildtype and D₁ or D₅ receptor knockout (KO) mice. Immunohistochemical and single-cell RT-PCR studies demonstrated a lack of compensatory effects after genetic deletion of D₁ or D₅ receptors. However, the expression of GABA_A receptor α1 subunits was reduced in D₅ KO mice. At the functional level, whole-cell patch clamp recordings in dissociated MSNs showed that GABA peak current amplitudes were smaller in cells from D₅ KO mice indicating that lack of this receptor subtype directly affected GABA_A-mediated currents. In striatal slices, addition of a D1 agonist reduced GABA currents significantly more in D₅ KO compared to D₁ KO mice. We conclude that D₁ receptors are the main D1-like receptor subtype involved in the modulation of GABA currents and that D₅ receptors contribute to the normal expression of these currents in the striatum. Special issue dedicated to Anthony Campagnoni.     

海藻希瓦氏菌(Shewanella alga)发酵培养基条件优化的研究

目的:优化海藻希瓦氏菌生产河豚毒素的发酵培养基。方法:通过测定菌体密度(用OD600表示)和菌体收获量,研究了部分初始条件及添加不同营养物质对海藻希瓦氏菌生长的影响,采用单因素试验和正交试验对发酵条件进行了优化。结果:最适发酵初始pH为7.5,最适摇瓶装液量为150mL。通过正交试验找出最大影响因素为葡萄糖供应,优化后的培养基最佳配方为:在2216E培养基中添加1.0%葡萄糖、2.5%酵母粉、1.0%磷酸高铁。结论:优化后的培养基培养供试菌,菌体收获量比在2216E培养基中培养增加了2.012g.L-1。     

Balance of Interactions Determines Optimal Survival in Multi-Species Communities

We consider a multi-species community modelled as a complex network of populations, where the links are given by a random asymmetric connectivity matrix J, with fraction 1 − C of zero entries, where C reflects the over-all connectivity of the system. The non-zero elements of J are drawn from a Gaussian distribution with mean μ and standard deviation σ. The signs of the elements J _ij reflect the nature of density-dependent interactions, such as predatory-prey, mutualism or competition, and their magnitudes reflect the strength of the interaction. In this study we try to uncover the broad features of the inter-species interactions that determine the global robustness of this network, as indicated by the average number of active nodes (i.e. non-extinct species) in the network, and the total population, reflecting the biomass yield. We find that the network transitions from a completely extinct system to one where all nodes are active, as the mean interaction strength goes from negative to positive, with the transition getting sharper for increasing C and decreasing σ. We also find that the total population, displays distinct non-monotonic scaling behaviour with respect to the product μC, implying that survival is dependent not merely on the number of links, but rather on the combination of the sparseness of the connectivity matrix and the net interaction strength. Interestingly, in an intermediate window of positive μC, the total population is maximal, indicating that too little or too much positive interactions is detrimental to survival. Rather, the total population levels are optimal when the network has intermediate net positive connection strengths. At the local level we observe marked qualitative changes in dynamical patterns, ranging from anti-phase clusters of period 2 cycles and chaotic bands, to fixed points, under the variation of mean μ of the interaction strengths. We also study the correlation between synchronization and survival, and find that synchronization does not necessarily lead to extinction. Lastly, we propose an effective low dimensional map to capture the behavior of the entire network, and this provides a broad understanding of the interplay of the local dynamical patterns and the global robustness trends in the network.     

杏鲍菇最佳培养基的筛选

研究了不同的母种、原种和栽培种培养基对杏鲍菇菌丝生长、子实体产量及生物学效率的影响。结果表明,杏鲍菇在不同配方的培养基上菌丝的生长存在明显差异,其中菌丝生长速度最快、最健壮的母种培养基为PDYA培养基;菌丝生长速度最快、子实体产量最高、生物学效率达91.7%的原种及栽培种培养基为配方2。原种及栽培种培养基的含水率为60%时,菌丝生长快而健壮,在生产上最适宜采用。     

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA_A receptors (GABA_ARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA_ARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA_AR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA_AR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA_ARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.     

Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.     

On the Occurrence of Spiny Vesicles in the Phloem of Salix

Many parenchyma cells in the primary phloem of young shootsof Salix contain spiny vesicles similar to those reported byNewcomb and others. They may occur singly or clustered, usuallyintermingled with other cell components. A lightly-stainingfibrous-granular background substance is generally found intheir vicinity. In most cells this substance is evenly dispersedin the cytoplasm; less frequently, a presumably similar substanceshowing a tendency to form compact clumps occurs in the vicinityof the spiny vesicles. These clumps occasionally show a tubularorganization, suggesting P-protein. The spiny vesicles are alsofound in association with well developed P-protein bodies insome cells. However, the question as to whether spiny vesiclesare connected with the formation of P-protein is left open.     

Network Bursting Dynamics in Excitatory Cortical Neuron Cultures Results from the Combination of Different Adaptive Mechanism

In the brain, synchronization among cells of an assembly is a common phenomenon, and thought to be functionally relevant. Here we used an in vitro experimental model of cell assemblies, cortical cultures, combined with numerical simulations of a spiking neural network (SNN) to investigate how and why spontaneous synchronization occurs. In order to deal with excitation only, we pharmacologically blocked GABA_Aergic transmission using bicuculline. Synchronous events in cortical cultures tend to involve almost every cell and to display relatively constant durations. We have thus named these “network spikes” (NS). The inter-NS-intervals (INSIs) proved to be a more interesting phenomenon. In most cortical cultures NSs typically come in series or bursts (“bursts of NSs”, BNS), with short (∼1 s) INSIs and separated by long silent intervals (tens of s), which leads to bimodal INSI distributions. This suggests that a facilitating mechanism is at work, presumably short-term synaptic facilitation, as well as two fatigue mechanisms: one with a short timescale, presumably short-term synaptic depression, and another one with a longer timescale, presumably cellular adaptation. We thus incorporated these three mechanisms into the SNN, which, indeed, produced realistic BNSs. Next, we systematically varied the recurrent excitation for various adaptation timescales. Strong excitability led to frequent, quasi-periodic BNSs (CV∼0), and weak excitability led to rare BNSs, approaching a Poisson process (CV∼1). Experimental cultures appear to operate within an intermediate weakly-synchronized regime (CV∼0.5), with an adaptation timescale in the 2–8 s range, and well described by a Poisson-with-refractory-period model. Taken together, our results demonstrate that the INSI statistics are indeed informative: they allowed us to infer the mechanisms at work, and many parameters that we cannot access experimentally.     

多花黄精诱导愈伤组织与不定芽培养基筛选

以多花黄精Polygonatum cyrtonema的根状茎为外植体,通过L9(34)正交试验比较不同植物生长调节剂及其组合对多花黄精愈伤组织诱导的影响,筛选最适生长调节剂配方,同时筛选通过器官发生方式直接成芽较为合适的培养基。结果表明,含有2,4-D和KT的培养基诱导愈伤组织效果显著,多花黄精愈伤组织诱导的最适培养基为MS + 6-BA 2.0 mg·L-1 + 2,4-D 0.5 mg·L-1 + NAA 0.1 mg·L-1 + KT 1.0 mg·L-1,该培养基的愈伤组织诱导率可达39.10%;培养基MS + 6-BA 4.0 mg·L-1 + NAA 0.2 mg·L-1可诱导多花黄精根状茎直接产生不定芽。