Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.     

Early development of voltage-dependent sodium currents in cultured mouse spinal cord neurons

Spinal cord neurons were dissociated from 13-day embryonic mice and grown in culture for 1-28 days. Sodium currents of neurons in culture for 1-2 days were compared with those in culture for 2-4 weeks, using the whole-cell voltage clamp method. Rapid neurite outgrowth created space clamp limitations so that unclamped neuritic sodium action potentials prevented accurate analysis of sodium current properties. Therefore neurons were bathed in sodium-free solution and brief puffs of sodium were delivered to the cell soma so that only somatic sodium currents were recorded. Sodium currents of neurons at 1-2 days in culture had voltage-dependent activation and inactivation characteristic of these channels, both in mature cultured spinal neurons and in other preparations. However, the estimated channel density on the soma of neurons 1-2 days in culture was less than two channels per micron2. Since the available sodium conductance (as measured by action potential rise rates) increases during development of spinal cord neurons in culture (Westbrook and Brenneman, 1984), we suggest that changes in channel density and/or distribution, rather than in channel kinetics, may underlie the increase in sodium conductance.     

Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: correlation with survival factor requirements in culture

The presence of nerve growth factor receptors and the imipramine-sensitive uptake of catecholamines in sympathetic neurons of chick embryos were investigated by autoradiography. Neurons were dissociated from paravertebral ganglia of different embryonic ages and receptors and catecholamine uptake were then determined both at the beginning of culture and after 2 days in culture, at which time the number of surviving neurons is determined by the survival factors present. It was found that while essentially all the neurons specifically bound 125I-NGF both after dissociation and at the end of the culture period, only 60% of the neurons take up [3H]norepinephrine after dissociation, and this proportion remained constant through the culture period under conditions where all the neurons survived. All of the neurons maintained by NGF in culture (35% of the total) displayed this uptake, and in contrast, only one-quarter of those maintained by heart cell-conditioned medium alone (60% of the total) took up catecholamines. The uptake was shown to be neither induced by NGF nor suppressed by heart cell-conditioned medium. These results support the hypothesis that chick sympathetic ganglia contain discrete subpopulations of neurons which may be selected in culture by virtue of their different requirements for survival factors.     

Locust primary neuronal culture for the study of synaptic transmission

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.     

Expression of somatostatin and GABA immunoreactivity in cultures of rat hippocampus

Somatostatin (SOM) synthesis and release were studied with radioimmunoassay and immunocytochemical techniques in rat fetal hippocampal neurons maintained in monolayer tissue culture. SOM immunoreactivity increased from undetectable to over 4,000 pg/ml in media and over 2,500 pg/culture in neurons by 3 to 5 weeks. After 3 weeks, approximately 11% of the neurons stained for SOM. Gamma-aminobutyric (GABA) immunoreactivity was present in hippocampal neurons from 1 day to 5 weeks with 40-50% of the neurons staining for GABA by 5 weeks in vitro. Costaining neurons for SOM and GABA revealed that 63% which were positive for SOM also stained for GABA.     

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.     

A neurite-promting factor from muscle supports the survival of cultured chicken spinal motor neurons

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%–6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutininhorseradish peroxidase or the fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.     

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.     

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Sensitivity of Aplysia neurons in primary culture to putative neurotransmitters

Aplysia neurons grown in primary cell culture (Dagan and Levitan, 1981) were exposed to the putative neurotransmitters acetylcholine and serotonin by local iontophoretic application, and changes in membrane potential or voltage clamp currents were examined. It was found that 47% of the neurons were sensitive to cholinergic agonists, 14% to serotonin, and 9% responded to both. Responses could be recorded upon application of the transmitters to the cell bodies as well as along the regenerated neurites. An identified group of neurons, the neurosecretory bag cells, exhibited similar responses to cholinergic agonists in culture and in situ. Pleural medial neurons exhibited cholinergic responses in culture similar to those previously reported in situ. Thus neurotransmitter receptor/ion channel complexes characteristic for a specific cell type in the intact ganglion are also present on this cell type in culture.     

Effects of nerve growth factor on the development of the dendritic system of cholinergic neurons in dissociated culture of the rat septum]

Neurons dissociated from septal area of fetal (E18-19) rat brain were grown 14-days in culture. Cholinergic neurons were identified by cytochemical demonstration of acetyl cholinesterase. It was shown that the nerve growth factor added to the culture medium (50 u/ml) has increased the size of cell body of AchE-positive neurons, mean total length and arborization of dendrites and also the dendritic tree area.     

Differential Ca(2+) signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture

Physiological studies on functionally identified myenteric neurons are scarce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca(2+) concentration ([Ca(2+)](i)) signaling to study excitatory neurotransmitter responsiveness of myenteric motor neurons. 1, 1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea pig ileum. DiI-labeled neurons were easily detectable in cultures prepared from these segments. The excitatory neurotransmitters (10(-5) M) acetylcholine, substance P, and serotonin induced a transient rise in [Ca(2+)](i) in subsets of DiI-labeled neurons (66.7, 56.5, and 84. 3%, respectively). DiI-labeled motor neurons were either inhibitory (23.8%) or excitatory (76.2%) as assessed by staining for nitric oxide synthase or choline acetyltransferase. Compared with excitatory motor neurons, significantly fewer inhibitory neurons in culture responded to acetylcholine (0 vs. 69%) and substance P (12.5 vs. 69.2%). We conclude that combining retrograde labeling and Ca(2+) imaging allows identification of differential receptor expression in functionally identified neurons in culture.     

Effect of K+- and kainate-mediated depolarization on survival and functional maturation of GABAergic and glutamatergic neurons in cultures of dissociated mouse cerebellum

The effect of the depolarizing agents, an elevated potassium concentration (25 mM) or kainic acid (50 μM) on neuronal survival and differentiation was investigated in cultures of dissociated neurons from cerebella of 7-day-old mice. When maintained in the presence of an antimitotic agent such cultures consist primarily of glutamatergic and GABAergic neurons. Cell survival was monitored by measurement of DNA, and differentiation by determining uptake and depolarization coupled release of glutamate (D-aspartate as label) and GABA. The depolarizing agents were added separately or together either from the start of the culture period (7–8 days) or at day 5 in culture. The main findings are that K⁺ depolarization is important for differentiation of glutamatergic neurons but not for GABAergic neurons. This depolarizing signal is important during the early phase of development in culture. For glutamatergic neurons, kainate may replace K⁺ as a depolarizing signal whereas in case of the GABAergic neurons, kainate was toxic particularly during the late phase of development. It was further observed that the glutamatergic neurons when maintained in a medium with 5 mM K⁺ during the first 5 days in culture became sensitive to kainate toxicity when this amino acid was added at day 5. This was not the case when the medium contained 25 mM K⁺ from the start of the culture period. Special issue dedicated to Dr. Kinya Kuriyama.     

Characterization of a co-culture system of neurons and hepatocytes

A co-culture system of cerebellar granule cells (glutamatergic neurons) and hepatocytes has been developed. Petri dishes divided in halves by a temporary septum were coated with poly-L-lysine and cerebellar granule cells plated in one of the compartments. Five days later hepatocytes were plated in the other compartment and after 2 days the septum was removed and the two cell types shared the same culture medium for a period of 5 days. During this period of time cultures of neurons and hepatocytes kept separately or in co-culture exhibited identical characteristics with regard to activities of pyruvate kinase and glucokinase (hepatocytes), aspartate aminotransferase (neurons) as well as evoked transmitter release (neurons) and content of cytochrome P-450 (hepatocytes). The results show that it is possible to maintain neurons and hepatocytes in co-culture sharing the same culture medium for a prolonged period of time. Such a system may serve as a pharmacological model to study interactions between liver and brain cells with regard to neuroactive drugs.     

No evidence for effects of mild microwave irradiation on electrophysiological and morphological properties of cultured embryonic rat dorsal root ganglion cells

Effects of mild microwave treatment (1 hr, 37 degrees C) on the in vitro development of rat mechanically dissociated dorsal root ganglion (DRG) neurons were investigated to establish whether microwave irradiation effects exist on nervous tissue other than heat induced tissue fixation. Phase contrast microscopy and immunocytochemical neurofilament stainings did not reveal significant differences between irradiated (2 hr after isolation) and control cultures, maintained up till 21 days. The electrophysiological properties of microwave exposed and non-exposed DRG neurons were compared using the whole-cell patch-clamp technique. Control neurons, in culture for 0-12 days, were excitable. In cultured cells (1-12 days), microwaved 2 hr after isolation, the action potentials were similar to or slightly different from those of the control cells. No acute microwave effects were found on neurons irradiated after 1 day of culture. These results suggest that mild microwave irradiation has neither significant acute nor strong long-term effects on DRG culture development and DRG neuron membrane properties, consistent with the notion that microwave effects essentially are temperature effects.     

无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。