Interaction of nicotinic acetylcholine receptors with dopamine receptors in synaptic plasticity of the mouse insular cortex

The insular cortex plays essential roles in nicotine addiction. However, much is still unknown about its cellular and synaptic mechanisms responsible for nicotine addiction. We have previously shown that in layer 5 pyramidal neurons of the mouse insular cortex, activation of the nicotinic acetylcholine receptors (nAChRs) suppresses synaptic potentiation through enhancing GABAergic synaptic transmission, although it enhances both glutamatergic and GABAergic synaptic transmission. In the present study, we examined whether dopamine receptors might contribute to the nicotine‐induced inhibition of synaptic potentiation. The nicotine‐induced inhibition of synaptic potentiation was decreased in the presence of a D1 dopamine receptor antagonist SCH23390 irrespective of the presence of a D2 dopamine receptor antagonist sulpiride, suggesting that D1 dopamine receptors are involved in nicotine‐induced inhibition. We also investigated how dopamine receptors might contribute to the nAChR‐induced enhancement of glutamatergic and GABAergic synaptic transmission. The nAChR‐induced enhancement of GABAergic synaptic transmission was decreased in the presence of SCH23390 irrespective of the presence of sulpiride, whereas that of glutamatergic synaptic transmission was not altered in the presence of SCH23390 and sulpiride. These results suggest that D1 dopamine receptors are involved in the nAChR‐induced enhancement of GABAergic synaptic transmission while dopamine receptors are not involved in that of glutamatergic synaptic transmission. These observations indicate that the interaction between nAChRs and D1 dopamine receptors plays critical roles in synaptic activities in layer 5 pyramidal neurons of the mouse insular cortex. These insular synaptic changes might be associated with nicotine addiction.     

大鼠皮质神经元的体外培养和纯化

目的：探讨大鼠皮质神经元分离纯化的有效方法。方法：取胎鼠大脑皮质细胞，分别在不同的培养液中进行原代培养，利用相差显微镜对培养的细胞进行动态观察；并以免疫细胞化学技术对神经元进行染色。结果：在相差显微镜下可见加用N2添加剂和胎牛血清培养的神经元生长良好，胞体形态多样，突起数目多，长度长，与邻近神经元的胞体或突起形成接触，而胶质细胞则大量死亡，神经元纯化率达94％以上，未加用N2添加剂培养的神经元胶质细胞大量存在，神经元纯化率达50％左右。结论：N2添加剂和胎牛血清联合应用可使大鼠皮质神经元得到良好的生长和有效纯化。     

补阳还五汤对大鼠脑皮层神经元生长的影响

目的观察含补阳还五汤的药物血清对体外培养大鼠脑皮层神经元生长的影响,为补阳还五汤治疗缺血性脑损伤提供实验依据。方法以体外培养新生SD乳鼠大脑皮层神经元为观察对象,采用四唑盐(MTT)比色法测定细胞生长情况。观察含药血清对常规培养及在缺氧状态下神经元生长的影响。结果补阳还五汤对常规培养及在缺氧条件下培养的神经元有显著促进生长作用,与空白血清组比较有显著差(P<0.05)。结论大鼠以补阳还五汤灌胃给药后,其血清中的药物成份有促进体外培养神经元细胞生长的作用。     

Morphology of corticotectal cells in the primary visual cortex of hooded rats

In primary visual cortex of hooded rats, pyramidal cells in layer V may be classified as long, medium, or short, on the basis of the layer in which the apical dendrite terminates. The present study determines which of these types of pyramidal cells project to the superior colliculus. Two different strategies were used to label corticotectal cells with horseradish peroxidase (HRP). In the first set of experiments, a large number of corticotectal cells were labeled by retrograde transport following injection of HRP into the superior colliculus. In the second set of experiments, single unit recording was used to identify corticotectal cells physiologically by antidromic activation from the superior colliculus. These cells were then impaled and labeled by intracellular iontophoresis of HRP. The results from both techniques suggest that only long pyramidal cells send an axon to the superior colliculus. These cells are distinguished by an apical dendrite that extends into layer I. We conclude that in hooded rats corticotectal cells in primary visual cortex are the long pyramids in layer V.     

pp60c-src encoded by the proto-oncogene c-src is a product of sensory neurons

The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes. One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina. Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene. This antiserum recognizes pp60c-src specifically in normal chicken cells. Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src. Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity. Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons. Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, all pp60c-src-positive cells were immunoreactive with neurofilament antibodies. pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones. These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture.     

In vivo Intracellular Recording of Neurons in the Supraoptic Nucleus of the Rat Hypothalamus

Intracellular recordings were made from cells in the hypothalamic supraoptic nucleus in the urethane-anaesthetized male rat using the ventral surgical approach. Impalements lasted from 5 min to 1 h and recorded cells had an input resistance of 55 to 170 megohms. Spikes of over 50 mV were recorded from 14 cells which could be antidromically activated by stimulation of the neural stalk. The spikes showed a hyperpolarizing afterpotential and the broadening characteristic of rapidly firing magnocellular neurons, which recovered rapidly (<200 ms). When depolarized, the cells showed evidence of a transient potassium current. Recurrent synaptic coupling between the recorded cell and adjacent cells would be expected to alter the hyperpolarizing afterpotential of an antidromic spike as compared with a spontaneous spike; no perceptible difference in the waveforms of the different types of spike could be detected in 11 spontaneously active cells. Application of just subthreshold stimuli to the neural stalk did not evoke depolarizing or hyperpolarizing potentials. Suprathreshold shocks to the neural stalk, when the antidromic spike was prevented by collision, also had no discernible effect on membrane potential. Thus intracellular recordings from magnocellular neurons in vivo revealed electrophysiological properties similar to those seen in vitro. No evidence for synaptic interconnection between magnocellular neurons was found in male rats.     

Chemically distinct rat olivocochlear neurons.

We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203-214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD-positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP-positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP-positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems.     

Synaptic organization of cortico-cortical connections from the primary visual cortex to the posteromedial lateral suprasylvian visual area in the cat

The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.     

Cat-301 immunoreactivity in the lateral geniculate nucleus and visual cortex of the strabismic amblyopic cat

Purpose: It was investigated whether alterations in neuronal structure and function occasioned by strabismic amblyopia also may be reflected in alterations in the expression on Y type neurons of a Cat-301 antibody sensitive antigen in the lateral geniculate nucleus (LGN) and cortex of our cat model of strabismic amblyopia. Methods/Results: The percentage of positively labelled cells was reduced in LGN laminae that received input from the deviated eye in strabismic amblyopic cats compared with normal cats. In the strabismic cortex, the density of immunopositive neurons was significantly reduced compared with normal, the effect being most pronounced in layer IV Conclusions: Despite previous physiological recordings indicating a decrease in X-cell associated acuity in strabismic amblyopia, the present findings imply that the changes in the early visual experience occasioned by strabismus also produce specific molecular changes in theY neuronal class.     

谷氨酸诱导体外培养的鸡胚脊髓神经细胞释放NO

用鸡胚脊髓神经细胞的原代培养,测定细胞中亚硝酸盐的含量,研究了谷氨酸(Glu)对原代培养神经细胞中NO的影响。结果表明,谷氨酸作用于原代培养的鸡胚脊髓神经细胞,在诱导神经细胞释放乳酸脱氢酶(LDH)的同时,还可诱导细胞释放一氧化氮(NO)。如先用NO合成酶抑制剂L-NOARG作用细胞,再加入Glu,则发现L-NOARG能降低Glu导致的培养液中LDH活性升高。提示NO可能参与介导Glu的神经毒性作用。