Srdija Jeftinija

ATP stimulates calcium-dependent glutamate release from cultured astrocytes.

ATP caused a dose‐dependent, receptor‐mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P2 receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca2+‐EGTA saline, suggesting the involvement of intracellular calcium stores. Pre‐treatment of glial cultures with an intracellular Ca2+ chelator abolished the stimulatory effects of ATP. Thapsigargin (1u2003µm), an inhibitor of Ca2+‐ATPase from the Ca2+ pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10u2003µm), a phospholipase C inhibitor, attenuated the ATP‐stimulatory effect on calcium transients and blocked ATP‐evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP‐induced glutamate release. Furthermore, inhibition of the glutamate transporters p‐chloromercuri‐phenylsulfonic acid and ltrans‐pyrolidine‐2,4‐dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl− channel blocker, 5‐nitro‐2(3‐phenylpropylamino)‐benzoate, reduced ATP‐induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium‐dependent anion‐transport sensitive mechanism.

Synergistic Effects of Physical and Chemical Guidance Cues on Neurite Alignment and Outgrowth on Biodegradable Polymer Substrates

This article demonstrates that directional outgrowth of neurites is promoted by applying a combination of physical and chemical cues to biodegradable polymer substrates. Films of poly-D,L-lactic acid and poly(lactide-co-glycolide) were micropatterned to form grooves on substrate surfaces, using novel indirect transfer techniques developed specifically for biodegradable polymers that cannot be micropatterned directly. Laminin was selectively adsorbed in the grooves. Whole and dissociated dorsal root ganglia were seeded on the substrates and neurite outgrowth and alignment along the microgrooves were measured. The microgrooves provide physical guidance, whereas laminin provides chemical cues to the neurons. The groove depth and spacing were found to significantly influence neurite alignment. The presence of laminin was found to promote neurite adhesion and outgrowth along the grooves. Using a combination of optimized physical and chemical cues, excellent spatial control of directional neurite outgrowth, with up to 95% alignment of neurites, was obtained. The synergistic effect of physical and chemical guidance cues was found to be more effective than individual cues in promoting directional outgrowth of neurites.

The role of tetrodotoxin-resistant sodium channels of small primary afferent fibers

Intracellular recordings from neurons in the dorsal root ganglion (DRG) and dorsal horn (DH), in an in vitro spinal cord-dorsal root ganglion preparation, were used to investigate the role of tetrodotoxin-resistant (TTX-R) afferent fibers in the sensory synaptic transmission in the superficial DH. Bath application of 25-50 mM potassium to the DRG depolarized the DRG neurons, blocked action potentials in the large neurons, evoked action potentials in slow conducting neurons, and synaptically excited dorsal horn neurons. Excitatory postsynaptic potentials (EPSP) which were evoked in DH neurons by electrical stimulation of large myelinated fibers, but not those evoked by stimulation of small unmyelinated fibers, were blocked by the potassium treatment of the primary afferents. Tetrodotoxin, when applied to the sensory neurons, abolished the action potentials in fast fibers but had no effect on the action potentials in a population of slow conducting afferents. Peripheral application of TTX blocked the fast EPSPs evoked by electrical stimulation but failed to block the electrically evoked slow EPSPs and the synaptic activation of DH neurons induced by the application of high potassium to sensory neurons. Furthermore, high potassium potentiated electrically evoked, TTX-resistant EPSPs in the majority of neurons. This effect was abolished in Na(+)-free solution. These findings indicate that high [K+]e applied to the DRG, dorsal root and peripheral process selectively activates a primary afferent input to the DH, which is sodium-dependent and tetrodotoxin resistant.

Micropatterned Schwann cell-seeded biodegradable polymer substrates significantly enhance neurite alignment and outgrowth.

Biomimetic strategies were employed to promote directional outgrowth of neurites in vitro by using a synergistic combination of physical, chemical, and cellular cues. Compression molded and solvent cast biodegradable polymer substrates made of poly(D,L-lactic acid) were micropatterned to form grooves on the substrate surfaces. Laminin was localized in the grooves, and rat sciatic Schwann cells were seeded on the substrates. Whole as well as dissociated rat dorsal root ganglia were seeded on the substrates along with Schwann cells, and neurite outgrowth and alignment were measured. The micropatterns provide physical guidance, laminin provides chemical cues, and the Schwann cells provide biological cues to the axons. The presence of Schwann cells in the grooves was found to promote neurite alignment as well as outgrowth and help the neurites orient even on shallower grooves and exhibit continued alignment even as the grooves degrade. The synergistic combination of physical, chemical, and cellular guidance enabled greater than 98% alignment of neurites and accelerated outgrowth of neurites in the direction of the microgrooves.

Cultured astrocytes express proteins involved in vesicular glutamate release

Bradykinin induces receptor-mediated calcium-dependent release of glutamate from cultured astrocytes through a mechanism that is neither due to cell-swelling mechanism nor due to the reversal of the glutamate transporter. Astrocytes may thus release glutamate using a mechanism resembling the neuronal vesicular release of neurotransmitters. Synaptobrevin is a vesicular protein that together with plasma membrane proteins syntaxin and SNAP-25 participate in formation of the anchoring core complex required for initiation of exocytosis. Here, we demonstrate that synaptobrevin II is present in cultured astrocytes. Furthermore, we demonstrate that botulinus toxin type B and tetanus toxin cause a decrease in synaptobrevin II immunoreactivity and abolish bradykinin-induced release of glutamate from cultured astrocytes. While we were not able to demonstrate the presence of SNAP-25 or syntaxin immunoreactivity in cultured astrocytes, pretreatment with BoTx-A (which cleaves SNAP-25) and BoTx-C (which cleaves syntaxins) result in a decrease in the baseline release of glutamate and diminish the bradykinin-evoked release of glutamate from cultured astrocytes. These findings strongly support the notion that astrocytes may release neurotransmitters using a mechanism similar to the neuronal secretory process.

Synergistic effects of micropatterned biodegradable conduits and Schwann cells on sciatic nerve regeneration

This paper describes a novel biodegradable conduit that provides a combination of physical, chemical and biological cues at the cellular level to facilitate peripheral nerve regeneration. The conduit consists of a porous poly(D,L-lactic acid) (PDLLA) tubular support structure with a micropatterned inner lumen. Schwann cells were pre-seeded into the lumen to provide additional trophic support. Conduits with micropatterned inner lumens pre-seeded with Schwann cells (MS) were fabricated and compared with three types of conduits used as controls: M (conduits with micropatterned inner lumens without pre-seeded Schwann cells), NS (conduits without micropatterned inner lumens pre-seeded with Schwann cells) and N (conduits without micropatterned inner lumens, without pre-seeded Schwann cells). The conduits were implanted in rats with 1 cm sciatic nerve transections and the regeneration and functional recovery were compared in the four different cases. The number or size of regenerated axons did not vary significantly among the different conduits. The time of recovery, and the sciatic function index, however, were significantly enhanced using the MS conduits, based on qualitative observations as well as quantitative measurements using walking track analysis. This demonstrates that biodegradable micropatterned conduits pre-seeded with Schwann cells that provide a combination of physical, chemical and biological guidance cues for regenerating axons at the cellular level offer a better alternative for repairing sciatic nerve transactions than conventional biodegradable conduits.

Neuroligand-Evoked Calcium-Dependent Release of Excitatory Amino Acids from Cultured Astrocytes

Abstract: The release of excitatory amino acids (EAAs) from neuron‐free cultures of neocortical astrocytes was monitored using HPLC. The neuroligand bradykinin caused a dose‐dependent receptor‐mediated increase in release of the EAAs glutamate and aspartate from type 1 astrocyte cell cultures obtained from rat cerebral cortex. Removal of calcium from the extracellular fluid prevented the bradykinin‐induced release of EAAs from astrocytes. The addition of the calcium ionophore ionomycin caused a calcium‐dependent release of EAAs. Inhibitors of the glutamate transporters p‐chloromercuriphenylsulfonic acid, l‐trans‐pyrrolidine‐2,4‐dicarboxylate, and dihydrokainate failed to impair the ability of bradykinin to stimulate glutamate release from astrocytes. α‐Latrotoxin, an active compound of black widow spider venom, caused a significant increase of the release of glutamate in calcium‐containing saline. In calcium‐depleted saline, α‐latrotoxin produced an initial increase in the concentration of glutamate followed by a decline in the concentration of glutamate indicating stimulation of exocytosis coupled with low calcium‐induced inhibition of endocytosis. Taken together, these data suggest that astrocytes may release neurotransmitter through a mechanism that is similar to the neuronal secretory process. Given the important role of glutamate in the induction of long‐term potentiation, learning, memory, and excitotoxicity, it will be important to determine external signals that control both the uptake and release of glutamate by astrocytes.

Excitatory amino acids are released from rat primary afferent neurons in vitro

Multiple lines of evidence implicate the excitatory amino acids (EAAs) (L-aspartate (L-Asp) and L-glutamate (L-Glu) as excitatory transmitters in the spinal cord. The specific objective of this study was to determine whether the EAAs are released from primary afferents. Dorsal root ganglia (DRG) from 2 to 18-day-old rats dissected and cultured for 1-2 weeks were washed in modified Ringers recording solution for a period of 1 h to allow equilibration. The mean +/- S.E.M. baseline concentrations of EAAs recovered during a 5 min interval were 533.29 +/- 65.59 nmol for L-Glu and 106.67 +/- 14.05 nmol for L-Asp. Stimulation of DRG organotypic cultures with potassium resulted in a significant concentration-dependent increase in the release of both EAAs. The concentration of Asp increased to 166 +/- 17% and 203 +/- 13% in response to 5 min exposure of the culture to 25 and 50 mM potassium, respectively. The concentration of Glu increased to 155 +/- 12% and 226 +/- 18% of control in response to the same stimuli. In response to application of 50 mM potassium for 25 min, peak concentrations increased to 465 +/- 53% for Asp and 312 +/- 51% for Glu of the basal concentration. Exposure of the cultures to 1 or 10 microM capsaicin also caused release of both EAAs. The concentrations of Asp and Glu significantly increased to 204 +/- 11% and 165 +/- 15% of basal concentrations, respectively, in response to a 5 min exposure to 1 microM capsaicin. High [K+]e failed to increase the release of EAAs from cultures where DRG cell bodies were removed 72 h prior to release experiments. These results confirm results demonstrating release of EAA from mammalian spinal cord tissues and directly demonstrate for the first time that primary afferent fibers are specifically involved in this release.

α-Latrotoxin stimulates glutamate release from cortical astrocytes in cell culture

The mechanism responsible for the ability of bradykinin to cause calcium‐dependent release of glutamate from astrocytes in vitro was investigated. The glutamate transport inhibitor, dihydrokainate, did not block bradykinin‐induced glutamate release, and bradykinin did not cause cell swelling. These data exclude the involvement of glutamate transporters or swelling mechanisms as mediating glutamate release in response to bradykinin. α‐Latrotoxin (3 nM), a component of black widow spider venom, stimulated calcium‐independent glutamate release from astrocytes. Since α‐latrotoxin induces vesicle fusion and calcium‐independent neuronal neurotransmitter release, our data suggest that astrocytes may release neurotransmitter using a mechanism similar to the neuronal secretory process.

Enkephalins modulate excitatory synaptic transmission in the superficial dorsal horn by acting at μ-opioid receptor sites

The effect of opioids on synaptic potentials of dorsal horn (DH) neurons has been investigated in a rat spinal cord DH slice-dorsal root ganglion (DRG) in vitro preparation. Conventional intracellular recording from DH and DRG neurons using 3 M potassium acetate-filled electrodes was employed. Dorsal roots were electrically isolated from the spinal cord slice and stimulated with pulses of different intensity and duration to evoke afferent action potentials monitored intracellularly from DRG neurons. Low-intensity single-shock stimulation of the dorsal roots (8-20 V pulses of 0.02-0.05 ms duration) activated large primary afferents and elicited excitatory postsynaptic potentials (EPSP) in all of the neurons tested. High-intensity stimulation of the dorsal roots (over 35 V pulses of 0.5 ms duration), sufficient to excite small myelinated and unmyelinated primary afferents resulted in a large and prolonged depolarization of DH neurons associated with firing of action potentials. Bath application (D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin (DAGO), (D-Ala2,D-Leu5)-enkephalinamide (DADLEA), or (D-Ala2,D-Met5)-enkephalinamide (DADMEA) produced dose-dependent, reversible hyperpolarization in about 75% of the neurons tested. The hyperpolarization was associated with a fall in neuronal input resistance. In addition, opioids depressed the synaptic transmission in all of the neurons examined. This depressant effect of opioids was independent from their effects on resting membrane potential. Delta specific receptor opioid agonists (D-Pen2.5)-enkephalin (DPDPE) and (D-Pen2,L-Pen5)-enkephalin (DPLPE), were completely ineffective in producing an effect on neuronal membrane or synaptic transmission. All opioid effects were antagonized by naloxone.