Isolated Rat Spinal Dorsal Horn Research Articles

Intracellular calcium homeostasis changes induced in rat spinal cord neurons by extracellular acidification.

Changes in intracellular Ca2+ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca2+]i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca2+]i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca2+ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca2+]i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca2+ channels, also substantially alters the Ca2+ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.

Orphanin FQ/nociceptin modulates glutamate- and kainic acid-induced currents in acutely isolated rat spinal dorsal horn neurons

The heptadecapeptide orphanin FQ (OFQ), also known as nociceptin (NOC), is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, the effects of OFQ/NOC on glutamate (Glu), kainic acid (KA) and quisqualic acid (QA) induced currents were examined in isolated rat spinal dorsal horn neurons of young rats using whole-cell patch-clamp techniques. Glu, KA and QA elicited rapid inward currents in 90%, 69%, 83% of tested neurons. OFQ/NOC(0.03∼300 nM) failed to induce any changes of membrane currents, but modulated Glu-, KA- and QA-elicited currents. OFQ/NOC inhibited and potentiated Glu-induced currents in 40.6% and 27.3% of examined cells (n=106) respectively. In about one third examined neurons, OFQ/NOC had no detectable effects on Glu responses. OFQ/NOC also inhibited and enhanced KA- and QA-induced currents (inhibition: KA, 67.1%, n=76; QA, 50%, n=36. Potentiation: KA, 23.7%, n=76; QA, 16.7%, n=36). In about 10% of tested cells, Gluinduced currents were potentiated after the application of OFQ/NOC, and lasted for 20∼30 min. The inhibitory effects of OFQ/NOC on KA and QA responses were naloxone-insensitive. The C-terminal fragment OFQ(8–17) presented same effects on EAA-induced responses. Taken together, OFQ/NOC primarily inhibited Glu-, KA- and QA-induced currents in isolated rat spinal dorsal horn neurons via non-opioid mechanism, which might contribute to nociceptive transmission in the spinal level.

Alpha-subunit of CaM-KII increases glycine currents in acutely isolated rat spinal neurons.

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the glycine receptor can be regulated by exogenous calcium/ calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced glycine receptor-activated current recorded with the use of the whole cell patch-clamp technique. This result suggests that the function of glycine receptor is modulated by CaM-KII, but the cellular mechanism underlying the enhancement of glycine receptor-activated current is still unknown.

Alpha-subunit of calcium/calmodulin-dependent protein kinase II enhances gamma-aminobutyric acid and inhibitory synaptic responses of rat neurons in vitro.

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the gamma-aminobutyric acid-A (GABAA) receptor can be regulated by calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced GABAA-receptor-activated current recorded with the use of the whole cell patch-clamp technique. This effect was associated with reduced desensitization of GABA responses. 2. GABA-induced currents are also potentiated by calyculin A, an inhibitor of protein phosphatases 1 and 2A. 3. Conventional intracellular recordings were made from hippocampal CA1 neurons in slices to determine the effect of intracellular application of CaM-KII on inhibitory synaptic potentials evoked by electrical stimulation of the stratum oriens/alveus. The inhibitory synaptic potential was enhanced by CaM-KII; this mechanism may contribute to long-term enhancement of inhibitory synaptic transmission and may also play a role in other forms of plasticity in the mammalian brain.

Alpha subunit of calcium/calmodulin-dependent protein kinase enhances excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

1. Here we report that in acutely isolated rat spinal dorsal horn (DH) neurons, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptors can be regulated by endogenous and exogenous calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced AMPA/kainate and NMDA currents recorded with the use of the whole cell patch-clamp technique. 2. Microcystin, a nonselective phosphatases inhibitor, also enhances AMPA and NMDA responses. 3. Conventional intracellular recordings were made from substantia gelatinosa neurons in spinal cord slices to determine the effect of intracellular application of CaM-KII on excitatory synaptic potentials evoked by electrical stimulation of primary afferent fibers. Excitatory synaptic transmission was enhanced by CaM-KII, which is consistent with the importance of phosphorylation of the postsynaptic AMPA/kainate and NMDA receptor-ion complexes in the short- and long-term changes in synaptic transmission.

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 n M )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.

Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid

In freshly isolated spinal dorsal horn (DH) neurons (laminae I–IV) of the young rat the effects of 25–100 μM of (±)- trans-1-aminocyclopentane-1,3-dicarboxylic acid ( trans-ACPD), 1 S,3 R-ACPD and 1 R,3 S-ACPD, a metabotropic glutamate receptor (mGluR) agonist, on inward currents induced by glutamate (Glu), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N- methyl- d-aspartate (NMDA) and kainate were studied under whole-cell voltage-clamp conditions. When the cells were clamped to −60 mV, the racemic mixture and both stereo isomers of trans-ACPD increase the responses elicited by Glu, AMPA, and NMDA, but little those of kainate. In addition, quisqualate (10–50 μM), in the presence of CNQX (5–20 μM) or NBQX (5 μM), potentiated NMDA-induced currents. The enhancing effect lasted 10–75 min, depending upon both dose and length of application. In a smaller proportion of dorsal horn neurons, the enhancing effect was preceded by a transient depression of the responses to Glu, AMPA, and NMDA. 2-Amino-3-phosphonopropionic acid (L-AP 3), a putative antagonist of mGluR exerted little effect on responses to AMPA itself, but reduced or prevented the enhancing effect of 1 S,3 R-ACPD. It is concluded that activation of a metabotropic glutamate receptor by trans-ACPD, and its two enantiomers, may mediate the enhancement of AMPA and NMDA responses in acutely isolated rat spinal dorsal horn neurons. These results are consistent with the possibility that the activation of metabotropic glutamate receptor may contribute to the regulation of the strength of excitatory amino-mediated primary afferent neurotransmission, including nociception.

Modulation of NMDA-induced currents by mu-opioid receptor agonist DAGO in acutely isolated rat spinal dorsal horn neurons.

The whole-cell patch-clamp technique was used to examine effects of mu-opioid receptor agonist Tyr-D-Ala-Gly-Me-Phe-Gly-ol-enkephalin (DAGO) on N-methyl-D-aspartate (NMDA)-induced currents in freshly enzymatically and/or mechanically dissociated rat spinal dorsal horn neurons (laminae I-III). Here we report that the responses of dorsal horn neurons to NMDA were modulated by DAGO in a complex manner. When applied simultaneously with, or prior to NMDA, DAGO (10-100 nM) initially suppressed the response to NMDA in 52% of examined cells. Following removal of DAGO, the NMDA responses were potentiated in 71% of the cells. The enhancing effect of DAGO was rapid in onset and lasted up to 50 min after removal of the peptide. Both the initial depressant and the late enhancing effect were reversed by naloxone. These results are consistent with the possibility that DAGO might directly modulate the NMDA receptor-ion channel complex and that this action may contribute to the regulation of the strength of excitatory amino acid-mediated primary afferent neurotransmission, including nociception.

Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones

The whole-cell patch-clamp technique was used to examine the effect of substance P (SP) on glutamate-induced currents in freshly dissociated rat spinal dorsal horn neurons (LI-III). In 48% of examined cells SP (10 −10–10 −6 M) at −70 mV, induced an inward current that desensitized in the continued presence of SP. When applied simultaneously with, or prior to l-glutamate, SP caused a potentiation of l-glutamate-induced current in 65% of the tested cells. Since glutamate activates both N- methyl- d-aspartate (NMDA) and non-NMDA receptors in rat dorsal horn neurons, selective agonists, kainate, quisqualate, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and NMDA were used to determine which subtype of excitatory amino acid receptors interacted with SP. We found that the responses to quisqualate, kainate, and AMPA were not significantly affected by SP (< 20% increase). In contrast, the inward currents induced by NMDA (30–300 μM) appear to be reduced and potentiated after the administration of 2–200 nM of SP. These results suggest that post-synaptic mechanisms of action of tachykinins may contribute to the regulation of the strength of glutamate-mediated excitatory transmission in the rat spinal dorsal horn.

Modulation of glutamate responses in isolated rat spinal dorsal horn neurons by substance P

Modulation of glutamate responses in isolated rat spinal dorsal horn neurons by substance P

Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons

The responses to excitatory and inhibitory amino acids and peptides were investigated in isolated rat spinal dorsal horn neurons (laminae I–V) of young rats using the whole-cell voltage-clamp technique. The treatment of spinal slices with low concentrations of enzymes and mechanical dissociation yielded isolated neurons that were sensitive to excitatory amino acids (glutamate, kainate, quisqualate and N- methyl- d-aspartate (NMDA), inhibitory amino acids (γ-aminobutyric acid (GABA), glycine) and peptides (substance P, calcitonin gene-related peptide (CGRP). The responses of dorsal horn neurons to NMDA were potentiated by glycine and CGRP, whereas GABA A responses were enhanced by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Our observations indicate that there is reasonable agreement between many of the responses of isolated neurons and those studied in in vivo and in vitro slice and culture preparations.