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A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF3) showed potent inhibition of MC activity with an EC50of 24.5 μM. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABABreceptor blocker prevented the KRS-5Me-4-OCF3-evoked inhibitory effects. In the presence of GABAAreceptor antagonists, KRS-5Me-4-OCF3completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF3-induced inhibition may be mediated by direct action on GABAAreceptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF3on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABAAreceptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF3, demonstrating that KRS-5Me-4-OCF3enhanced GABA affinity and acted as a positive allosteric modulator of GABAAreceptors. The effect of KRS-5Me-4-OCF3was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF3binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF(3)) showed potent inhibition of MC activity with an EC(50) of 24.5 μM. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABA(B) receptor blocker prevented the KRS-5Me-4-OCF(3)-evoked inhibitory effects. In the presence of GABA(A) receptor antagonists, KRS-5Me-4-OCF(3) completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF(3)-induced inhibition may be mediated by direct action on GABA(A) receptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF(3) on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABA(A) receptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF(3), demonstrating that KRS-5Me-4-OCF(3) enhanced GABA affinity and acted as a positive allosteric modulator of GABA(A) receptors. The effect of KRS-5Me-4-OCF(3) was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF(3) binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

The main olfactory bulb receives a significant modulatory noradrenergic input from the locus coeruleus. Previous in vivo and in vitro studies showed that norepinephrine (NE) inputs increase the sensitivity of mitral cells to weak olfactory inputs. The cellular basis for this action of NE is not understood. The goal of this study was to investigate the effect of NE and noradrenergic agonists on the excitability of mitral cells, the main output cells of the olfactory bulb, using whole cell patch-clamp recording in vitro. The noradrenergic agonists, phenylephrine (PE, 10 μM), isoproterenol (Isop, 10 μM), and clonidine (3 μM), were used to test for the functional presence of α1-, β-, and α2-receptors, respectively, on mitral cells. None of these agonists affected olfactory nerve (ON)–evoked field potentials recorded in the glomerular layer, or ON-evoked postsynaptic currents recorded in mitral cells. In whole cell voltage-clamp recordings, NE (30 μM) induced an inward current (54 ± 7 pA, n= 16) with an EC50of 4.7 μM. Both PE and Isop also produced inward currents (22 ± 4 pA, n= 19, and 29 ± 9 pA, n= 8, respectively), while clonidine produced no effect (n= 6). In the presence of TTX (1 μM), and blockers of excitatory and inhibitory fast synaptic transmission [gabazine 5 μM, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 μM, and (±)-2-amino-5-phosphonopentanoic acid (APV) 50 μM], the inward current induced by PE persisted (EC50= 9 μM), whereas that of Isop was absent. The effect of PE was also observed in the presence of the Ca2+channel blockers, cadmium (100 μM) and nickel (100 μM). The inward current caused by PE was blocked when the interior of the cell was perfused with the nonhydrolyzable GDP analogue, GDPβS, indicating that the α1 effect is mediated by G-protein coupling. The current-voltage relationship in the absence and presence of PE indicated that the current induced by PE decreased near the equilibrium potential for potassium ions. In current-clamp recordings from bistable mitral cells, PE shifted the membrane potential from the downstate (−52 mV) toward the upstate (−40 mV), and significantly increased spike generation in response to perithreshold ON input. These findings indicate that NE excites mitral cells directly via α1 receptors, an effect that may underlie, at least in part, increased mitral cell responses to weak ON input during locus coeruleus activation in vivo.

Synaptic mechanisms underlying NMDA-mediated responses of neurons in the guinea pig lateral amygdala (AL) were investigated in in vitroslice preparations. Local application of NMDA resulted in initial hyperpolarization of pyramidal-like spiny cells (projection neurons), followed by prolonged depolarization. The slow depolarization represented a direct postsynaptic effect of NMDA, whereas the initial hyperpolarization was induced presynaptically through activation of GABAergic interneurons and was sensitive to blockade by tetrodotoxin as well as the GABAA-receptor antagonist bicuculline. Application of NMDA resulted in AP-5-sensitive, lasting depolarization also in putative interneurons of the AL suggesting direct activation of GABAergic interneurons by NMDA. These data indicate that interneurons in the rat lateral amygdala possess functional NMDA receptors, which may contribute to the predominantly inhibitory synaptic responses in amygdaloid neurons following activation through afferent input systems.

By means of intracellular recording and staining, we studied the ability of several distinct classes of projection (output) neurons, which innervate the sexually dimorphic macroglomerular complex (MGC‐PNs) in the antennal lobe of the male moth Manduca sexta, to encode naturally intermittent sex pheromonal stimuli. In many MGC‐PNs, antennal stimulation with a blend of the two essential pheromone components evoked a characteristic triphasic response consisting of a brief, hyperpolarizing inhibitory potential (I1) followed by depolarization with firing of action potentials and then a delayed period of hyperpolarization (I2). MGC‐PNs described in this study resolved pulsed pheromonal stimuli, up to about five pulses/second, with a distinct burst of action potentials for each pulse of odor. The larger the amplitude of I1, the higher the pulse rate an MGC‐PN could follow, illustrating the importance of inhibitory synaptic input in shaping the temporal firing properties of these glomerular output neurons. In some MGC‐PNs, triphasic responses were evoked by antennal stimulation with only one of the two key pheromone components. Again, the maximal pulse rate that an MGC‐PN could follow with that pheromone component as sole stimulus was high in MGC‐PNs that responded with a strong I1. These component‐specific MGC‐PNs innervated only one of the two principal glomeruli of the MGC, while MGC‐PNs that were primarily excited by antennal stimulation with either key pheromone component had arborizations in both major MGC glomeruli. These observations therefore suggest that the population of antennal olfactory receptor cells responding to a single pheromone component is functionally heterogeneous: a subset of these sensory cells activates the excitatory drive to many uniglomerular MGC‐PNs, while others feed onto inhibitory circuits that hyperpolarize the same PNs. This convergence of opposing inputs is a circuit property common to uniglomerular MGC‐PNs branching in either of the major MGC glomeruli, and it enhances the ability of these glomerular output neurons to resolve intermittent olfactory input. Synaptic integration at the uniglomerular PN level thus contributes to the transmission of behaviorally important temporal information about each key pheromone component to higher centers in the brain. J. Comp. Neurol. 409:1–12, 1999. © 1999 Wiley‐Liss, Inc.

Adenosine is a depressant in the central nervous system with pre‐ and postsynaptic effects. In the present study, intracellular recording techniques were applied to investigate the modulatory effects of adenosine on projection neurons in the lateral rat amygdala (LA), maintained as slices in vitro.Adenosine reversibly reduced the amplitude of a fast inhibitory postsynaptic potential (IPSP) that was evoked by electrical stimulation of the external capsule and pharmacologically isolated by applying an N‐methyl‐D‐aspartate and non‐N‐methyl‐D‐aspartate receptor antagonist, DL‐(−)‐2‐amino‐5‐methyl‐4‐isoxazolepropionic acid and 6,7‐Dinitroquinoxaline‐2,3‐dione, respectively, and the γ‐aminobutyric acidB(GABAB) receptor antagonist CGP 35348. The postsynaptic potential that remained was abolished by locally applying bicuculline.Adenosine reduced the amplitude of the fast IPSP on average by 40.3%. It had no significant effect on responses to exogenously applied GABA, on membrane potential or on input resistance, suggesting that the site of action was at presynaptic inhibitory interneurons in the LA.The response to adenosine was mimicked by the selective adenosine A1receptor agonist N6‐cyclohexyladenosine and blocked by the selective adenosine A1receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine.Neuronal responsiveness in the amygdala is largely controlled by inhibitory processes. Adenosine can presynaptically downregulate inhibitory postsynaptic responses and could exert dampening effects likely by depression of both excitatory and inhibitory neurotransmitter release.