• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.283-292
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• 1996

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region. In this study, single neurons of the spinal dorsal horn were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by patch clamp technique. Transverse slice ($(300{\mu}m$) of lumbar spinal cords from young rats$(7{\sim}14\;days)$ were sequentially treated with two pretenses(pronase 0.75 mg/ml and thermolysin 0.75 mg/ml), then single neurons were mechanically dissociated. These neurons showed near-intact morphology such as multipolar, ellipsoidal and bipolar, and pyramidal cells and we recorded the typical whole cell currents of $K^+$, $Ca^{2+}$ and ligand-operated channels from these neurons. Glutamate $(30{\mu}M)$ and N-methyl-D-aspartate(NMDA, $30{\mu}M)$ induced inward currents of $117{\pm}12.4$ pA(n=5) and $49{\pm}6.9$ pA(n=3), respectively. Glycine $(1{\mu}M)$ potentiated glutamate-induced currents $4{\sim}5$ times and NMDA-induced currents $8{\sim}10$ times. In addition, glycine $(30{\mu}M)$ induced Inward current ($31{\pm}6.1$ nA, n=2), which was rapidly desensitized after the peak to a new steady-state level. However, the inward currents induced by ${\gamma}-amino$ butyric acid(GABA, $1{\mu}M$) decreased continuously after the peak($226{\pm}41.6$ pA, n=3) under the similar experimental condition. The ionic currents and pharmacological responses of isolated neurons in this work were similar to those observed in vivo or in vitro spinal cord slice, indicating that acutely isolated neurons could be effectively used for further pharmacological studies.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.175-175
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• 1996

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is Processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region and sites and cellular mechanisms of their actions have been a target of numerous studies. In this study, single neurons were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by the patch clamp method. Young rats (7-14 days) were anesthetized with diethyl-ether, and the lumbar spinal cord was excised and cut transversely at a thickness of 30$\mu\textrm{m}$ by Vibroslicer. The treatment of spinal slices with low concentration of proteases (pronase and thermolysin 0.75 mg/$m\ell$) and mechanical dissociation yielded isolated neurons with near intact morphology. Multipolar, ellipsoidal and bipolar, and pyramidal cells were shown. By applying step voltage pulses to neurons held at -70 mV, two types of inward currents and one outward currents observed. The fast activating and inactivating inward current was the Na$\^$+/ current because of its fast kinetics and blocking by 0.5${\mu}$M TTX, a specific blocker of Na$\^$+/ channel. The second type of inward currents were sustained. Based on their kinetics and current-voltage relations, it was likely that the second type of inward current was the voltage-dependent Ca$\^$2+/ current. In the presence of TTX, the steady-state currents mainly represented outward K$\^$+/ current which looked like the delayed rectifier K$\^$+/ current. In addition, the membrane currents produced by agonist of excitatory amino acid (EAA) receptor and the endogenous transmitter candidate L-glutamate were recorded in isolated whole-cell voltage clamped neurons as well as responses to inhibitory amino acids (${\gamma}$-amino butyric acid, glycine). Drugs were applied by a method that allows complete exchange of the solution within 1 sec; an infinite number of solutions can be applied to a single cell.