Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

The Xiro-repressed gene CoREST is expressed in Xenopus neural territories.

The iroquois (iro) genes encode evolutionary conserved homeoproteins that participate in many developmental processes [reviewed in Development 128 (2001) 2847]. In Xenopus, the Iro protein Xiro1 is a repressor, required during gastrulation for neural plate formation, that downregulates Bmp4. During neurulation, Xiro1 participates in the pattering of the neuroectoderm. In this work, we report the cloning and pattern of expression of XCoREST, another gene repressed by Xiro1. During Xenopus development, XCoREST is expressed in territories in which neurogenesis takes place.     

弱噪声对小鼠下丘神经元频率调谐的影响

为探讨弱噪声对小鼠 (MusmusculusKm)中脑下丘 (inferiorcolliculus ,IC)神经元声信号提取的影响 ,采用单位胞外记录方法 ,研究了加入弱白噪声 (强度相当于纯音阈强度下 5dB)前后神经元频率调谐曲线的变化。实验共记录到 10 4个下丘神经元 ,测量了 32个神经元的频率调谐曲线。结果显示 :①弱噪声条件下神经元的频率调谐曲线表现出 3种类型 ,即锐化 (34 4 % ,11/ 32 )、拓宽 (18 8% ,6 / 32 )和不受影响 (4 6 9% ,15 / 32 ) ,其中锐化呈现有意义的变化 ;②频率调谐受弱噪声锐化的神经元 ,其Q10 、Q3 0 平均分别增大 (34 4 2±17 0 4 ) % (P =0 0 2 6 ,n =11)和 (4 6 34± 2 2 88) % (P =0 0 0 9,n =7) ,且Q3 0 变化率大于Q10 ;③弱噪声对调谐曲线的高、低频边锐化度不一 ,神经元低频边的反转斜率基本不变 [由 0 16± 0 0 8变为 0 16± 0 0 7kHz/dB (P =0 94 7,n =7) ],而高频边明显下降 [由 0 5 2± 0 2 5下降为 0 2 6± 0 13kHz/dB ,平均减小 (4 3 81±2 4 0 6 ) % ,(P =0 0 4 6 ,n =7) ]。上述结果表明 ,弱噪声可锐化小鼠IC神经元频率调谐 ,并强化神经元的声信号高频分析能力     

Calcium signal‐dependent plasticity of neuronal excitability developed postnatally

Neuronal plasticity and its development were investigated at pyramidal neurons in the cortical slices of rats. The threshold and probability of firing spikes were measured by using whole‐cell recording to assess neuronal excitability. Postsynaptic high frequency activity (HFA) at the pyramidal neurons, evoked by 20 trains (250‐ms interval) of five depolarization‐pulses (1 ms) at 100 Hz, persistently lowered the threshold and increased the probability of firing spikes. After long‐term enhancement of neuronal excitability by HFA was stable, another HFA induced further enhancement. Infusing 1 mM 1,2‐bis(2‐aminophenoxy)‐ethane‐N, N,N′,N′‐tetraacetic acid or 100 μM CaMKII(281–301) into the recording neurons prevented HFA‐induced long‐term enhancement of neuronal excitability. The infusion of 40 μM calcineurin autoinhibitory peptide enhanced neuronal excitability, which occluded HFA effect. HFA‐induced long‐term enhancement of intrinsic excitability expressed at most pyramidal neurons after postnatal day (PND) 14, but not at those before PND 9. Our results show a new type of neuronal plasticity induced by physiological activity at cortical neurons, which requires calcium‐dependent protein phosphorylation and develops during postnatal period. An upregulation of intrinsic excitability at cortical neurons facilitates their activity and broadens signal codes; consequently, their computational ability is upgraded. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Neuronal Organization of the Telencephalon in the Chum Salmon Onchorhynchus keta

This paper presents original data on the neuronal composition of various regions of the pallium of the telencephalon in Oncorhynchus keta. This study was carried out using routine neurohistologic techniques. Four basic areas were distinguished within the pallium: medial, dorsal, central, and lateral. The central pallium exhibited the most complicated cytoarchitectonics. In the central and lateral areas, pyramidal-like neurons with a well-developed dendritic spine apparatus were found. These were allodendritic cells, which appeared to be comparable to the pyramidal neurons of higher vertebrates in a number of features. Horizontal neurons and isodendritic radial neurons were also encountered. These cell types occurred in the ventral part of the central area and in the lateral area.     

Evolution and development of time coding systems

Precise temporal coding is a hallmark of both the electrosensory and auditory systems. Selective pressures to improve accuracy or encode more rapid changes have produced a suite of convergent physiological and morphological features that contribute to temporal coding. Comparative studies of temporal coding can also point to shared computational strategies, and suggest how selection might act to improve coding.     

温度对下丘脑神经元膜上延迟整流性K~ 通道活动密度的影响

目的 :探讨温度对下丘脑神经元延迟整流性K 单离子通道 (Ik)活动密度的影响。方法 :采用膜片钳细胞贴附式技术观察和记录 32℃、37℃和 39℃时下丘脑神经元Ik 的活动。结果 :温度升高 ,记录到封接膜片上Ik 活动的机率增大 ,由 32℃时的 34.6 %升至 39℃时的 5 1.0 % ,而且出现多通道活动 ;Ik 通道活动密度明显增高 (P <0 .0 5 ) ,32℃、37℃和 39℃时膜上的通道活动密度分别为 0 .71、1.2 2和 1.5 2channels/ μm2 。结论 :温度升高 ,下丘脑神经元上Ik 通道更多地处于活动状态 ,这可能有利于下丘脑神经元对体温活动的调节     

P2X2, P2X2–2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neurons

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.