Guo-Yuan Hu

Comparison of the effects of cholinesterase inhibitors on [3H]MK-801 binding in rat cerebral cortex.

Huperzine A, a selective inhibitor of acetylcholinesterase, was recently demonstrated to exert an antagonist effect on N-methyl-D-aspartate (NMDA) receptor in rat cerebral cortex. In the present study, the effects of six cholinesterase inhibitors, e.g. huperzine A, huperzine B, tacrine, donepezil (E2020), physostigmine and galanthamine on [3H]dizocilpine (MK-801) binding to synaptic membrane of rat cerebral cortex were compared. Their IC50 values (mean +/- SD) were 36.9 +/- 12.1, 316.8 +/- 93.2, 33.2 +/- 3.7, 135.0 +/- 15.1, 50.4 +/- 7.4, and 3344 +/- 295 microM, respectively. The rank order of potency is tacrine approximately huperzine A > physostigmine > donepezil > huperzine B >> galanthamine. There is no correlation between their activities to inhibit [3H]MK-801 binding and to inhibit acetylcholinesterase (r = +0.563, P = 0.245). The results suggest that most cholinesterase inhibitors available exhibit an antagonist effect on NMDA receptor in rat cerebral cortex in addition to their inhibitory effect on acetylcholinesterase.

Electrophysiological and morphological properties of pyramidal and nonpyramidal neurons in the cat motor cortex in vitro.

Electrophysiological and morphological properties of the neurons in cat motor cortex were investigated using intracellular recording and staining techniques in a brain slice preparation. In response to intracellular injection of depolarizing current pulses, four distinct types of firing patterns were observed among cat neocortical neurons. Regular-spiking neurons were characterized by their repetitive firing from which conspicuous frequency adaptation was observed. Doublet-or-burst firing cells were marked with their tendency to fire 2-5 clustered spikes at the onset of depolarizing pulse. In doublet-or-burst firing neurons, but not in regular-spiking neurons, a low-threshold calcium current was revealed by single-electrode voltage clamp. Both regular-spiking and doublet-or-burst firing neurons had relatively wide action potentials. Fast-spiking neurons could fire extremely narrow action potentials at a very high frequency. Their frequency-to-intensity slope of steady-state firing was significantly higher than that of the other neurons. In contrast, narrow-spiking neurons had the smallest frequency-to-intensity slope for steady-state firing, although their action potentials were as narrow as those of the fast-spiking neurons. Both regular-spiking and doublet-or-burst firing neurons were identified as pyramidal neurons, and were found in all layers below layer I. Their apical dendrites were densely coated with dendritic spines. Narrow-spiking neurons were only recorded in layer V. They were large pyramidal cells with scare spines on their apical dendrites. Fast-spiking neurons were all nonpyramidal interneurons. Seven out of eight labelled fast-spiking cells had beaded dendrites without spines. Their axons had a large number of varicosities, and arborized extensively to form a dense plexus of terminals in the vicinity of their soma. The remaining neuron was found to be a spiny nonpyramidal neuron in layer V. These results demonstrate that, in addition to the three types of firing patterns previously identified in rodent neocortex, a group of neurons in the cat motor cortex express another type of firing behaviour which is characterized by extremely narrow action potential and very small frequency-to-intensity slope. Correlation with the morphological data shows that these neurons are large layer V pyramidal cells rather than nonpyramidal interneurons.

Huperzine A, a nootropic alkaloid, inhibits N-methyl-D-aspartate-induced current in rat dissociated hippocampal neurons

Huperzine A, a nootropic alkaloid isolated from a Chinese herb, has been proposed as one of the most promising agents to treat Alzheimers disease. Recently, the agent was found to inhibit the N-methyl-D-aspartate (NMDA) receptors in rat cerebral cortex in addition to causing an inhibitory effect on acetylcholinesterase. In the present study, the mechanisms underlying NMDA receptor inhibition were investigated using whole-cell voltage-clamp recording in CA1 pyramidal neurons acutely dissociated from rat hippocampus. Huperzine A reversibly inhibited the NMDA-induced current (IC(50)=126 microM, Hill coefficient=0.92), whereas it had no effect on the current induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate or kainate. The effect was non-competitive, and showed neither voltage-dependency, nor use-dependency. The IC(50) values of huperzine A were neither altered by changing the concentrations of glycine (2-0.2 microM) and pH (7.4-6.7) in the external solution, nor by addition of Zn(2+) (5 microM) and dithiothreitol (5 mM) to the external solution. However, addition of spermine (200 microM) to the external solution caused a parallel shift to the right of the huperzine A concentration-response curve. From these we suggest that huperzine A acts as a non-competitive antagonist of the NMDA receptors, via a competitive interaction with one of the polyamine binding sites. The potential relevance of NMDA receptor antagonist activity of huperzine A to the treatment of Alzheimers disease is discussed.

Different mechanisms underlying the repolarization of narrow and wide action potentials in pyramidal cells and interneurons of cat motor cortex

Two different types of action potentials were observed among the pyramidal cells and interneurons in cat motor cortex: the narrow action potentials and the wide action potentials. These two types of action potentials had similar rising phases (528.8 +/- 77.0 vs 553.1 +/- 71.8 mV/ms for the maximal rising rate), but differed in spike duration (0.44 +/- 0.09 vs 1.40 +/- 0.39 ms) and amplitude (57.31 +/- 8.22 vs 72.52 +/- 8.31 mV), implying that the ionic currents contributing to repolarization of these action potentials are different. Here we address this issue by pharmacological manipulation and using voltage-clamp technique in slices of cat motor cortex. Raising extracellular K+ concentration (from 3 mM to 10 mM), applying a low dose of 4-aminopyridine (2-200 microM) or administering a low concentration of tetraethylammonium (0.2-1.0 mM) each not only broadened the narrow action potentials, but also increased their amplitudes. In contrast, high K+ medium or low dose of tetraethylammonium only broadened the wide action potentials, leaving their amplitudes unaffected, and 4-aminopyridine had only a slight broadening effect on the wide spikes. These results implied that K+ currents were involved in the repolarization of both types of action potentials, and that the K+ currents in the narrow action potentials seemed to activate much earlier than those in the wide spikes. This early activated K+ current may counteract the rapid sodium current, yielding the extremely brief duration and small amplitude of the narrow spikes. The sensitivity of the narrow spikes to 4-aminopyridine may not be mainly attributed to blockade of the classical A current (IA), because depolarizing the membrane potential to inactivate IA did not reproduce the effects of 4-aminopyridine. Blockade of Ca2+ influx slowed the last two-thirds repolarization of the wide action potentials. On the contrary, the narrow action potentials were not affected by Ca(2+)-current blockers, but if they were first broadened by 4-aminopyridine or tetraethylammonium, subsequent application of Ca(2+)-free medium caused further broadening, suggesting that the narrow action potentials were too brief to activate the Ca(2+)-activated potassium currents for their repolarization. Therefore, the effects of low concentrations of tetraethylammonium on the narrow spikes appeared to be mainly due to blockade of an outward current that was different from the tetraethylammonium-sensitive Ca(2+)-activated potassium current (IC). In the neurons with the narrow spikes, voltage-clamp experiments revealed two voltage-gated outward currents that were sensitive to tetraethylammonium and 4-aminopyridine, respectively. Both currents were activated rapidly following the onset of depolarizing steps. Interestingly, the tetraethylammonium-sensitive current was a transient outward current that inactivated rapidly (tau < or = 5 ms), while the 4-aminopyridine-sensitive current was relatively persistent during maintained depolarization. The 4-aminopyridine-sensitive current did not show obvious inactivation even at membrane potential of -40 mV, which completely inactivated the transient tetraethylammonium-sensitive, current. The results indicate that different potassium currents are involved in the repolarization of the narrow and wide action potentials in cat motor cortex. A novel tetraethylammonium-sensitive transient outward current and a 4-aminopyridine-sensitive outward current are responsible for the short duration and small amplitude of the narrow action potentials in the interneurons and some of the layer V pyramidal cells. These two currents are voltage-gated and Ca(2+)-independent. For the wide action potentials that characterize most pyramidal neurons, a Ca(2+)-independent tetraethylammonium-sensitive outward current and a Ca(2+)-activated potassium current are the main contributors to their repolarization.

Songorine, a diterpenoid alkaloid of the genus Aconitum, is a novel GABAA receptor antagonist in rat brain

Songorine, a diterpenoid alkaloid isolated from the genus Aconitum, was recently found to enhance the excitatory synaptic transmission in rat hippocampus. The mechanism underlying the effects was examined in the present study. The alkaloid at 0.1-300 microM inhibited the specific binding of [(3)H]muscimol to Triton-treated synaptic membranes of rat brain in a concentration-dependent manner (IC(50)=7.06 microM; 95% confidence limits: 3.28-10.84 microM). Scatchard analysis and Lineweaver-Burk double reciprocal plot of [(3)H]muscimol saturation binding data indicate a non-competitive inhibition of the alkaloid on the gamma-aminobutyric acid(A) (GABA(A)) receptor. In acutely dissociated rat hippocampal neurons the alkaloid did not elicit current response, but markedly inhibited the GABA-induced inward current (IC(50)=19.6 microM). The results suggest that songorine is a novel non-competitive antagonist at the GABA(A) receptor in rat brain.

Huperzine A, a nootropic agent, inhibits fast transient potassium current in rat dissociated hippocampal neurons

The actions of huperzine A (HupA), a novel cholinesterase inhibitor, on the fast transient potassium current (IA) were investigated in CA1 pyramidal neurons acutely dissociated from rat hippocampus. HupA reversibly inhibited IA (IC(50) = 914 +/- 1 microM). The effect was voltage-independent and insensitive to atropine. Tacrine was eight times more potent than HupA (IC(50) = 115 +/- 2 M), whereas huperzine B had little effect. HupA slowed down the decay of IA and its recovery from inactivation. HupA had no effect on the steady-state inactivation, but hyperpolarized the activation curve of IA by 6 mV. The results suggest that HupA may act as a blocker at the external mouth of the A channel. The potential relevance of the inhibitory effect of HupA on IA to the treatment of Alzheimers disease has been discussed.

Spermidine antagonizes the inhibitory effect of huperzine A on [3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex.

Huperzine A, a novel cholinesterase inhibitor, was found to inhibit the N-methyl-D-aspartate (NMDA) receptors in the brain. In this study, the mechanisms of the NMDA receptor inhibition were investigated using [3H]dizocilpine (MK-801) binding in synaptic membrane of rat cerebral cortex. Changing the concentrations of L-glutamate and L-glycine did not alter the potency of huperzine A. Spermidine caused rightward shift of the concentration-response curve of huperzine A, and considerably increased its IC(50) value. Huperzine A did not affect the potency of unlabeled (+)-MK-801 in [3H]MK-801 binding. Saturation binding studies reveal that huperzine A exerts a negative allosteric modulation on the MK-801 binding site within the NMDA receptor-channel. The results suggest that huperzine A is a non-competitive antagonist of the NMDA receptors, acting at one of the polyamine binding sites.

Similar potency of the enantiomers of huperzine A in inhibition of [3H]dizocilpine (MK-801) binding in rat cerebral cortex

The inhibition of huperzine A, a potential therapeutic agent to treat Alzheimers disease, on rat cortical acetylcholinesterase was found to be highly stereospecific. In the present study the effect of the enantiomers of huperzine A on [(3)H]dizocilpine (MK-801) binding to synaptic membrane of rat cerebral cortex was compared. The natural (-)-huperzine A and the synthetic (+)-huperzine A inhibited the specific binding of [(3)H]MK-801 with a similar potency. The IC(50) values were 65+/-7 and 82+/-12 microM (n=5 for each enantiomer, P=0.248), respectively. The result indicates that huperzine A inhibits N-methyl-D-aspartate (NMDA) receptor in rat cerebral cortex without stereoselectivity.

Huperzine A inhibits the sustained potassium current in rat dissociated hippocampal neurons

The actions of huperzine A (HupA), a novel cholinesterase inhibitor, on the sustained potassium current were investigated in acutely dissociated hippocampal neurons of rat. HupA inhibited the current (IC(50) = 856 +/- 1 microM) with voltage-dependency. The effect was insensitive to 3 microM atropine. Tacrine (IC(50) = 43 +/- 3 microM) was 20 times more potent than HupA. HupA hyperpolarized the activation curve of the current by 16 mV, and markedly prolonged the decay time constant tau(2). HupA affected neither the steady-state inactivation of the current, nor its recovery from inactivation. The potential relevance of the inhibitory effect of HupA on the current to the treatment of Alzheimers disease is discussed.

GABAA receptor-mediated feedback inhibition in pyramidal neurons of cat motor cortex

Feedback or recurrent inhibition is generally proposed as a basic component of circuit organization in the brain. However, there is little direct evidence for its existence in the cortex, nor for the nature of the neurotransmitters and receptors involved. Here we address this issue by analyzing the potentials following a single action potential in cat neocortical pyramidal neurons. Using 3 M KCl-filled electrodes, we observed a distinct depolarizing potential that was superimposed on the spike after-potentials in seven out of 81 recorded and the resemblance between its falling phase and the passive decay of membrane potential suggested that the depolarizing potential originated from the cell body or proximal dendrites. This potential was blocked by bicuculline methiodide (10 microM), but not by phaclofen (0.2 mM), suggesting that it was a reversed recurrent IPSP mediated by GABAA receptors.