A quantitative model of purinergic junctional transmission of calcium waves in astrocyte networks

A principal means of transmitting intracellular calcium (Ca2+) waves at junctions between astrocytes involves the release of the chemical transmitter adenosine triphosphate (ATP). A model of this process is presented in which activation of purinergic P2Y receptors by ATP triggers the release of ATP, in an autocrine manner, as well as concomitantly increasing intracellular Ca2+. The dependence of the temporal characteristics of the Ca2+ wave are shown to critically depend on the dissociation constant (K(R)) for ATP binding to the P2Y receptor type. Incorporating this model astrocyte into networks of these cells successfully accounts for many of the properties of propagating Ca2+ waves, such as the dependence of velocity on the type of P2Y receptor and the time-lag of the Ca2+ wave behind the ATP wave. In addition, the conditions under which Ca2+ waves may jump from one set of astrocytes across an astrocyte-free lane to another set of astrocytes are quantitatively accounted for by the model. The properties of purinergic transmission at astrocyte junctions may determine many of the characteristics of Ca2+ propagation in networks of these cells.     

Neuronal activity triggers calcium waves in hippocampal astrocyte networks.

The recent discovery that the neurotransmitter glutamate can trigger actively propagating Ca2+ waves in the cytoplasm of cultured astrocytes suggests the possibility that synaptically released glutamate may trigger similar Ca2+ waves in brain astrocytes in situ. To explore this possibility, we used confocal microscopy and the Ca2+ indicator fluo-3 to study organotypically cultured slices of rat hippocampus, where astrocytic and neuronal networks are intermingled in their normal tissue relationships. We find that astrocytic Ca2+ waves are present under these circumstances and that these waves can be triggered by the firing of glutamatergic neuronal afferents with latencies as short as 2 s. The Ca2+ waves closely resemble those previously observed in cultured astrocytes: they propagate both within and between astrocytes at velocities of 7-27 microns/s at 21 degrees C. The ability of tissue astrocyte networks to respond to neuronal network activity suggests that astrocytes may have a much more dynamic and active role in brain function than has been generally recognized.     

Optimized and efficient preparation of astrocyte cultures from rat spinal cord

Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1–2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G₂/M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.     

Purinergic signalling in a latent stem cell niche of the rat spinal cord

The ependyma of the spinal cord harbours stem cells which are activated by traumatic spinal cord injury. Progenitor-like cells in the central canal (CC) are organized in spatial domains. The cells lining the lateral aspects combine characteristics of ependymocytes and radial glia (RG) whereas in the dorsal and ventral poles, CC-contacting cells have the morphological phenotype of RG and display complex electrophysiological phenotypes. The signals that may affect these progenitors are little understood. Because ATP is massively released after spinal cord injury, we hypothesized that purinergic signalling plays a part in this spinal stem cell niche. We combined immunohistochemistry, in vitro patch-clamp whole-cell recordings and Ca²⁺ imaging to explore the effects of purinergic agonists on ependymal progenitor-like cells in the neonatal (P1–P6) rat spinal cord. Prolonged focal application of a high concentration of ATP (1 mM) induced a slow inward current. Equimolar concentrations of BzATP generated larger currents that reversed close to 0 mV, had a linear current–voltage relationship and were blocked by Brilliant Blue G, suggesting the presence of functional P2X7 receptors. Immunohistochemistry showed that P2X7 receptors were expressed around the CC and the processes of RG. BzATP also generated Ca²⁺ waves in RG that were triggered by Ca²⁺ influx and propagated via Ca²⁺ release from internal stores through activation of ryanodine receptors. We speculate that the intracellular Ca²⁺ signalling triggered by P2X7 receptor activation may be an epigenetic mechanism to modulate the behaviour of progenitors in response to ATP released after injury.     

Purinergic receptor signaling regulates N-cadherin expression in primary astrocyte cultures

Extracellular ATP exerts both short-term and long-term effects in the CNS by stimulating cell-surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N-cadherin expression, a calcium-dependent cell adhesion molecule involved in many processes, including glia-glia and axon-glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N-cadherin protein expression increased in a time- and concentration-dependent manner. In addition, ATP treatment caused an increase in N-cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N-cadherin to the cell surface membrane were independent of protein synthesis. The ATP-induced increase in N-cadherin protein expression was blocked by reactive blue 2 and 8-(p-sulfophenyl)-theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N-cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N-cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N-cadherin expression and suggest a role for this mechanism in cell-cell interactions.     

Local positive feedback by calcium in the propagation of intracellular calcium waves.

In many types of eukaryotic cells, the activation of surface receptors leads to the production of inositol 1,4,5-trisphosphate and calcium release from intracellular stores. Calcium release can occur in complex spatial patterns, including waves of release that traverse the cytoplasm. Fluorescence video microscopy was used to view calcium waves in single mouse neuroblastoma cells. The propagation of calcium waves was slowed by buffers that bind calcium quickly, such as BAPTA, but not by a buffer with slower on-rate, EGTA. This shows that a key feedback event in wave propagation is rapid diffusion of calcium occurring locally on a scale of < 1 micron. The length-speed product of wavefronts was used to determine that calcium acting in feedback diffuses at nearly the rate expected for free diffusion in aqueous solution. In cytoplasm, which contains immobile Ca2+ buffers, this rate of diffusion occurs only in the first 0.2 ms after release, within 0.4 micron of a Ca2+ release channel mouth. Calcium diffusion from an open channel to neighboring release sites is, therefore, a rate-determining regenerative step in calcium wave propagation. The theoretical limitations of the wave front analysis are discussed.     

Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.     

Purinergic receptors and synaptic transmission in enteric neurons

Purines such as ATP and adenosine participate in synaptic transmission in the enteric nervous system as neurotransmitters or neuromodulators. Purinergic receptors are localized on the cell bodies or nerve terminals of different functional classes of enteric neurons and, with other receptors, form unique receptor complements. Activation of purinergic receptors can regulate neuronal activity by depolarization, by regulating intracellular calcium, or by modulating second messenger pathways. Purinergic signaling between enteric neurons plays an important role in regulating specific enteric reflexes and overall gastrointestinal function. In the present article, we review evidence for purine receptors in the enteric nervous system, including P1 (adenosine) receptors and P2 (ATP) receptors. We will explore the role they play in mediating fast and slow synaptic transmission and in presynaptic inhibition of transmission. Finally, we will examine the molecular properties of the native receptors, their signaling mechanisms, and their role in gastrointestinal pathology.     

Nonlinear propagation of spherical calcium waves in rat cardiac myocytes.

Spontaneous calcium waves in enzymatically isolated rat cardiac myocytes were investigated by confocal laser scanning microscopy (CLSM) using the fluorescent Ca2+-indicator fluo-3 AM. As recently shown, a spreading wave of enhanced cytosolic calcium appears, most probably during Ca2+ overload, and is initiated by an elementary event called a "calcium spark." When measured by conventional fluorescence microscopy the propagation velocity of spontaneous calcium waves determined at several points along the cardiac myocyte was previously found to be constant. More precise measurements with a CLSM showed a nonlinear propagation. The wave velocity was low, close to the focus, and increased with increasing time and propagation length, approaching a maximum of 113 microns/s. This result was surprising, inasmuch as for geometrical reasons a decrease of the propagation velocity might be expected if the confocal plane is not identical with that plane where the focus of the wave was localized. It is suggested that the propagation velocity is essentially dependent on the curvature of the spreading wave. From the linear relationship of velocity versus curvature, a critical radius of 2.7 +/- 1.4 microns (mean +/- SD) was worked out, below which an outward propagation of the wave will not take place. Once released from a sufficiently extended cluster of sarcoplasmic reticulum release channels, calcium diffuses and will activate its neighbors. While traveling away, the volume into which calcium diffuses becomes effectively smaller than at low radii. This effect is the consequence of the summation of elementary events (Ca2+ sparks) and leads to a steeper increase of the cytosolic calcium concentration after a certain diffusion path length. Thus the time taken to reach a critical threshold of [Ca2+]i at the neighboring calcium release sites decreases with decreasing curvature and the wave will propagate faster.     

Partly shared spinal cord networks for locomotion and scratching

Animals produce a variety of behaviors using a limited number of muscles and motor neurons. Rhythmic behaviors are often generated in basic form by networks of neurons within the central nervous system, or central pattern generators (CPGs). It is known from several invertebrates that different rhythmic behaviors involving the same muscles and motor neurons can be generated by a single CPG, multiple separate CPGs, or partly overlapping CPGs. Much less is known about how vertebrates generate multiple, rhythmic behaviors involving the same muscles. The spinal cord of limbed vertebrates contains CPGs for locomotion and multiple forms of scratching. We investigated the extent of sharing of CPGs for hind limb locomotion and for scratching. We used the spinal cord of adult red-eared turtles. Animals were immobilized to remove movement-related sensory feedback and were spinally transected to remove input from the brain. We took two approaches. First, we monitored individual spinal cord interneurons (i.e., neurons that are in between sensory neurons and motor neurons) during generation of each kind of rhythmic output of motor neurons (i.e., each motor pattern). Many spinal cord interneurons were rhythmically activated during the motor patterns for forward swimming and all three forms of scratching. Some of these scratch/swim interneurons had physiological and morphological properties consistent with their playing a role in the generation of motor patterns for all of these rhythmic behaviors. Other spinal cord interneurons, however, were rhythmically activated during scratching motor patterns but inhibited during swimming motor patterns. Thus, locomotion and scratching may be generated by partly shared spinal cord CPGs. Second, we delivered swim-evoking and scratch-evoking stimuli simultaneously and monitored the resulting motor patterns. Simultaneous stimulation could cause interactions of scratch inputs with subthreshold swim inputs to produce normal swimming, acceleration of the swimming rhythm, scratch-swim hybrid cycles, or complete cessation of the rhythm. The type of effect obtained depended on the level of swim-evoking stimulation. These effects suggest that swim-evoking and scratch-evoking inputs can interact strongly in the spinal cord to modify the rhythm and pattern of motor output. Collectively, the single-neuron recordings and the results of simultaneous stimulation suggest that important elements of the generation of rhythms and patterns are shared between locomotion and scratching in limbed vertebrates.     

治疗脊髓损伤之建立星形胶质细胞模型的研究进展

脊髓损伤(spinal cord injury,SCI)是一种极为复杂的破坏性疾病,一旦脊髓损伤发生,治疗棘手,对患者家庭、国家带来巨大的经济、社会负担。近年来,通过建立大鼠脊髓损伤细胞相关模型,对于脊髓损伤的病因病机治疗等方面有了进一步的认识,而星形胶质细胞模型的建立对脊髓损伤治疗有深远意义。研究发现,星形胶质细胞作为靶细胞通过血-脑脊液屏障直接或间接对脊髓损伤有双向调控作用。本文通过对近年来星形胶质细胞模型培养制备方案等研究进行总结,以期为建立一个客观化、定量化、可模拟化的星形胶质细胞模型提供指导对脊髓损伤的治疗提供新的思路。     

A quantitative model of cortical spreading depression due to purinergic and gap-junction transmission in astrocyte networks

Spreading depression (SD), a propagating wave of electrical silence in the cortex and archicortex, involves depolarization of neurons and astrocytes for ∼1 min, due principally to a large increase in extracellular K⁺. SD is accompanied by large increases in extracellular ATP and is blocked by glutamate N-methyl-D-aspartate receptor antagonists. As a principal means of transmission between astrocytes is through their release of ATP, we have investigated if a model in which SD is driven by the effects of astrocyte waves of ATP interacting with waves of glutamate release from neurons and astrocytes can give a quantitative account of experimental observations on SD. We show that the characteristics of SD and the accompanying extracellular ionic changes can be accommodated by such a model—whether astrocyte transmission is principally through the release of ATP, as in archicortex (hippocampus) and spinal cord, or via gap junctions, as in the neocortex. Furthermore, these models give quantitative accounts of the effects on the characteristics of SD of agents toxic for astrocytes and of gap-junction blockers. Finally, an additional series of critical tests of the model is suggested.     

Estrogen alleviates neuropathic pain induced after spinal cord injury by inhibiting microglia and astrocyte activation

Neuropathic pain after spinal cord injury (SCI) is developed in about 80% of SCI patients and there is no efficient therapeutic drug to alleviate SCI-induced neuropathic pain. Here we examined the effect of estrogen on SCI-induced neuropathic pain at below-level and its effect on neuroinflammation as underlying mechanisms. Neuropathic pain is developed at late phase after SCI and a single dose of 17β-estradiol (100, 300?μg/kg) were administered to rats with neuropathic pain after SCI through intravenous injection. As results, both mechanical allodynia and thermal hyperalgesia were significantly reduced by 17β-estradiol compared to vehicle control. Both microglia and astrocyte activation in the lamina I and II of L4-5 dorsal horn was also inhibited by 17β-estradiol. In addition, the levels of p-p38MAPK and p-ERK known to be activated in microglia and p-JNK known to be activated in astrocyte were significantly decreased by 17β-estradiol. Furthermore, the mRNA expression of inflammatory mediators such as Il-1β, Il-6, iNos, and Cox-2 was more attenuated in 17β-estradiol-treated group than in vehicle-treated group. Particularly, we found that the analgesic effect by 17β-estradiol was mediated via estrogen receptors, which are expressed in dorsal horn neurons. These results suggest that 17β-estradiol may attenuate SCI-induced neuropathic pain by inhibiting microglia and astrocyte activation followed inflammation.     

Urotensin-II regulates intracellular calcium in dissociated rat spinal cord neurons

Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i increases in these neurons with a threshold of 10-9 m, and a maximal effect at 10-6 m with an estimated EC50 of 6.2 x 10-9 m. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10-7 m), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10-7 m) and the l-type Ca2+ channel blocker, verapamil (10-5 m) had minimal effects. Down-regulation of protein kinase C by 4-alpha-phorbol 12-myristate 13-acetate (10-6 m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10-6 m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10-6 m) or Rp-8-pCPT-cGMPS (3 x 10-5 m) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10-6 m) and Rp-cAMPS (3 x 10-5 m) reduced the response to 25 +/- 3% and 42 +/- 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.     

Cholinergic modulation of the synaptic transmission in the frog spinal cord

It was found during experiments on isolated frog spinal cord involving extracellular recording from the dorsal roots (sucrose bridging) and intracellular recording from motoneurons by microelectrodes that 10 mM of the M-cholinomimetic arecoline produces motoneuronal depolarization which is matched by depolarizing electronic ventral root potentials and a rise in motoneuronal input resistance. Arecoline changes synaptic transmission by increasing the amplitude of postsynaptic potentials during intracellular recording and that of motoneuronal reflex discharges in the ventral roots but reduces the duration of dorsal root potentials. In the presence of arecoline, L-glutamate-induced motoneuronal response increases. Facilitation of synaptic transmission produced by arecoline in the spinal cord is bound up with cholinergic M₂- activation, since it is suppressed by atropine but not by low concentrations of pirenzipine; it is also coupled with a reduction in adenylcyclase activity. When motoneuronal postsynaptic response has been suppressed, as in the case of surplus calcium or theophylline, arecoline produces an inhibitory effect on the amplitude of motoneuronal monosynaptic reflex discharges which is suppressed by pirenzipine at a concentration of 1×10^–7 M. This would indicate the presence at the primary afferent terminals of presynaptic cholinergic M₁ receptors which mediate its inhibition of impulses of transmitter release. This effect is independent of changes in cyclic nucleotide concentration.A. M. Gorkii Medical Institute, Donetsk. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 399–405, May–June, 1987.     

Interferon induces astrocyte maturation causing an increase in cholinergic properties of cultured human spinal cord cells

It has been well established that interferon-gamma (IFN-gamma) can modify the immune status of cells in the central nervous system (CNS) by inducing major histocompatibility antigens. Furthermore, it has been shown that endogenous IFN can be produced in the brain following viral infection and a form of IFN-alpha/beta can be produced by astrocytes in culture. Here we show that IFN can induce astrocyte maturation and alter neurotransmitter properties in cultured CNS neurons at a given developmental stage. IFN causes a dose-dependent increase in choline acetyltransferase activity and glial fibrillary acidic protein (GFAP) immunoreactivity in cultures of human embryonic spinal cord neurons. The GABAergic activity and the Thy1 immunoreactivity remain unchanged. IFN-gamma does not act directly on the neurons but via the nonneuronal cells, probably the astrocytes, which in turn stimulate the cholinergic traits. These studies could be important for demonstrating an action of the immune system on glial cell maturation and on the neurotransmitter phenotype expression in CNS neurons.