Odorant modulation of neuronal activity and local field potential in sensory-deprived olfactory bulb.

Neuronal discharge and local field potential (LFP) oscillations in the olfactory bulb (OB) are modulated by odorant stimulation. The LFP oscillations have been proposed as the mechanism that facilitates synchronization of OB output neurons and the representation of similar odorants. Gamma LFP oscillations depend on the OB inhibitory network and early sensory deprivation modifies this inhibitory network. However, little is known about the LFP oscillations and neuronal discharge in the deprived OB. We examined the mitral/tufted (MT) cells' oscillatory activity and LFP oscillations in both sensory-deprived and normal OBs in urethane anesthetized rats. We found that MT cells in deprived and normal OBs have similar basal mean firing rate; 44% of the recorded cells in deprived OB and only 8% of the cells in normal OB showed firing rate modulation by odorants, both exhibiting a similar ratio of excitatory to inhibitory responses. A fraction of MT cells exhibited oscillatory discharge centered on gamma (60-70 Hz) and beta (20 Hz) frequencies, although this feature was not consistently dependent on odorant stimulation. Odorants decreased the LFP oscillatory power in the gamma band (35-90 Hz) and increased the power in the beta band (12-30 Hz). The modulation of LFP oscillations by odorants was also predominant in the deprived (53%) compared to the normal OB (17%). In contrast, a higher fraction of MT cells' discharge was locked to the gamma LFP cycle in the normal OB. These results suggest that early unilateral olfactory deprivation increases the OB sensitivity to odorants and reduce the temporal synchrony between unitary activity and gamma LFP oscillations without altering the basal neuronal discharge.

Odor response properties of neighboring mitral/tufted cells in the rat olfactory bulb.

Olfactory perception initiates in the nasal epithelium wherefrom olfactory receptor neurons--expressing the same receptor protein--project and converge in two different glomeruli within each olfactory bulb. Recent evidence suggests that glomeruli are isolated functional units, arranged in a chemotopic manner in the olfactory bulb. Exposure to odorants leads to the activation of specific populations of glomeruli. In rodents, about 25-50 mitral/tufted cells project their primary dendrites to a single glomerulus receiving similar sensory input. Yet, little is known about the properties of neighboring mitral/tufted cells connected to one or a few neighboring glomeruli. We used tetrodes to simultaneously record multiple single-unit activity in the mitral cell layer of anesthetized, freely breathing rats while exposed to mixtures of chemically related compounds. First, we characterized the odorant-induced modifications in firing rate of neighboring mitral/tufted cells and found that they do not share odorant response profiles. Individual units showed a long silent (11.01 ms) period with no oscillatory activity. Cross-correlation analysis between neighboring mitral/tufted cells revealed negligible synchronous activity among them. Finally, we show that respiratory-related temporal patterns are dissimilar among neighboring mitral/tufted cells and also that odorant stimulation results in an individual modification that is not necessarily shared by neighboring mitral/tufted cells. These results show that neighboring mitral/tufted cells frequently exhibit dissimilar response properties, which are not consistent with a precise chemotopic map at the mitral/tufted cell layer in the olfactory bulb.

Non-NMDA and NMDA receptors in the synaptic pathway between area postrema and nucleus tractus solitarius.

Area postrema (AP) modulates cardiovascular function through excitatory projections to neurons in nucleus tractus solitarius (NTS), which also process primary sensory (including cardiovascular-related) input via the solitary tract (TS). The neurotransmitter(s) and their receptors in the AP-NTS pathway have not been fully characterized. We used whole cell recordings in voltage- and current-clamp modes in the rat brain stem slice to examine the role of ionotropic glutamatergic receptors and alpha2-adrenergic receptors in the pathway from AP to NTS neurons receiving visceral afferent information via the TS. In neurons voltage clamped at potentials from -100 to +80 mV, AP stimulation (0. 2 Hz) evoked excitatory postsynaptic currents having a fast component blocked by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxobenzoquinoxaline-7-sulfonamide (NBQX; 3 microM, n = 7) and a slow component blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 50 microM, n = 8). Although NBQX (3 microM, n = 14) abolished AP-evoked action potentials, APV (50 microM, n = 9 or 500 microM, n = 6) or yohimbine, (200 nM, n = 5 or 2 microM, n = 10) did not. Thus, although AP stimulation activates both non-NMDA and NMDA receptors on NTS neurons receiving TS input, only non-NMDA receptors are required for synaptic transmission.

NMDA receptors contribute to primary visceral afferent transmission in the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is a principal site for coordinating the reflex control of autonomic function. The nucleus receives and organizes primary visceral (sensory) afferent inputs from the great vessels, heart, lung, and gastrointestinal organs. Glutamate, the excitatory neurotransmitter released by the primary afferent fibers, activates non-N-methyl-D-aspartate (non-NMDA) receptors on second-order neurons in the NTS. Still in question is whether NMDA receptors on the second-order neurons are also activated. Accordingly, the purpose of this study was to directly determine whether NMDA receptors contribute to synaptic transmission of primary visceral afferent input to second-order neurons in the NTS. Whole cell patch-clamp recordings were obtained from intermediate and caudal NTS neurons in rat coronal medullary slices. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of the solitary tract (1-25 V, 0.1 ms, 0.2 or 0.5 Hz) at membrane potentials ranging from -90 to +60 mV. In 28 of 32 neurons in which current-voltage relationships were obtained for solitary-tract-evoked EPSCs, the currents had short onset latencies (3.42 +/- 1.03 ms, mean +/- SD), indicating that they were the result of monosynaptic activation of second-order neurons. Solitary-tract-evoked EPSCs had both a fast and a slow component. The amplitude of the slow component was nonlinearly related to voltage (being revealed only at membrane potentials positive to -45 mV), blocked by the NMDA receptor antagonist DL-2-amino-5-monophosphovaleric acid (APV, 50 microM; n = 12; P = 0.0001), and enhanced in nominally Mg2+-free perfusate at membrane potentials negative to -45 mV (n = 5; P = 0.016), demonstrating that the slow component was mediated by NMDA receptors. The amplitude of the fast component was linearly related to voltage and blocked by the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX, 3 microM; n = 9; P = 0.0014), demonstrating that the fast component was mediated by non-NMDA receptors. The slow component of the EPSCs was not blocked by NBQX (n = 6; P = 0.134), nor was the fast component blocked by APV (n = 12; P = 0.124). These results show that both NMDA and non-NMDA receptors coexist on the same second-order NTS neurons and mediate primary visceral afferent transmission in the NTS. The participation of NMDA receptors suggests that second-order neurons in the NTS may have previously unrecognized integrative capabilities in the reflex control of autonomic function.

Gating properties of mutant acetylcholine receptors.

A number of affinity labeling studies have identified several tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor as being in or near the ligand binding site. Studies employing site-directed mutagenesis of these residues (alpha Y93, alpha Y190, and alpha Y198; the notation used is subunit/amino acid/position in the Torpedo receptor/substitution) in mouse muscle, Torpedo electroplax, and alpha 7 neuronal acetylcholine receptors have demonstrated that substitution of phenylalanine for tyrosine results in a shift towards higher concentrations in the macroscopic dose-response curves for acetylcholine-elicited currents from voltage-clamped Xenopus oocytes that express the receptors. This decrease in apparent affinity has been ascribed to either a reduction in binding affinity or a reduction in the coupling of agonist binding to ion channel opening; both mechanisms would give rise to shifts in the dose-response curves. We have used kinetic analysis of ion channel gating at the single-channel level to obtain estimates for the rate constants associated with the ligand binding and channel opening steps for wild-type, alpha Y93F, and alpha Y198F receptors. The results suggest that the underlying cause of the shifts in the macroscopic dose-response curves is a reduction in acetylcholine affinity for the resting activatable state of the receptor. Furthermore, it is the association rate for agonist binding, rather than the dissociation rate, that is most affected by the mutations.

Ligand-receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the alpha subunit.

Affinity labeling studies have identified several conserved tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor (alpha Y93, alpha Y190, and alpha Y198) as being in or near the ligand binding site. Mutagenesis studies from several laboratories have shown that substitution of phenylalanine for tyrosine at these positions reduces the apparent affinity for ACh. We have examined this apparent reduction in affinity further through the use of multiple substitutions at each position. Substitution of either phenylalanine, tryptophan, or serine resulted in an apparent decrease in agonist affinity, but the degree of reduction depended on both the position and the nature of the substitution. Analysis of the effects of each substitution suggests that each residue interacts with the quaternary N of ACh, and that each residue may make a different type of interaction with the agonist.

Selective enhancement of the interaction of curare with the nicotinic acetylcholine receptor.

Alteration of the ligand-binding domain of the nicotinic acetylcholine receptor through site-directed mutagenesis offers a powerful approach to the elucidation of structure-function relations in the receptor. Several conserved tyrosine residues in the large extracellular amino terminus of the alpha subunit of the receptor have been implicated by both chemical labeling and mutagenesis studies as playing an important role in the interaction of acetylcholine with the receptor. We and others have previously shown that substitution of phenylalanine for tyrosine at position 198 of the alpha subunit (alpha Y198F) leads to a rightward shift in the dose-response curve for acetylcholine-elicited currents. We have further investigated this particular mutation by examining the interaction of the competitive antagonist d-tubocurarine (curare) with the receptor. In contrast to the effect on the interaction of agonists with the receptor, this mutation leads to a marked increase in the affinity of the receptor for curare. Furthermore, this enhancement in affinity is selective for curare and is not seen with other competitive antagonists (pancuronium, beta-erythroidine, and gallamine). Examination of the structures of these competitive antagonists leads to the proposal that this enhancement is due to the formation of an aromatic-aromatic interaction between the phenylalanine ring at position alpha 198 in the mutant and one of the aromatic rings of curare and that this can provide information about the spatial arrangement of this residue in the binding site.

Forskolin acts as a noncompetitive inhibitor of nicotinic acetylcholine receptors.

Bath application of micromolar concentrations of forskolin to Xenopus oocytes that express either Torpedo electroplax or mouse muscle nicotinic acetylcholine (ACh) receptors leads to a reduction in the size of the ACh-elicited currents. This inhibition is concentration dependent and rapidly reversible, with full onset and recovery occurring within the exchange time of the recording chamber. Torpedo and mouse ACh receptors exhibit differential sensitivity to forskolin, with the Torpedo receptor showing higher affinity than the mouse receptor, with Ki values of 6.5 microM and 22 microM, respectively. The affinity for forskolin increases with ACh concentration, which rules out the possibility that forskolin acts as a competitive inhibitor. Single-channel analysis using excised patches shows that forskolin has no effect on either the single-channel amplitude or mean open time but, instead, reduces the number of channel openings per unit time, suggesting that forskolin either is a very slow channel blocker or alters receptor gating such that a fraction of the channels enter a state from which they are no longer available to open. Finally, through the use of a series of mouse-Torpedo hybrid ACh receptors, it is shown that the structural features responsible for the observed species difference in the affinity of ACh receptors for forskolin, and thus at least part of the binding site, are located on the gamma subunit.

Niflumic and flufenamic acids are potent reversible blockers of Ca2(+)-activated Cl- channels in Xenopus oocytes.

The effects of niflumic acid and flufenamic acid, two nonsteroidal anti-inflammatory agents known to block anion transport in red blood cells, on Ca2(+)-activated Cl- currents (ICl(Ca)) in Xenopus oocytes were examined. Both compounds reversibly inhibited ICl(Ca), elicited in response to depolarizing voltage steps, in a dose-dependent manner, with no effect on the shape of the current-voltage curve. The apparent inhibition constant for niflumic acid was 17 microM, whereas that for flufenamic acid was 28 microM. Niflumic acid also inhibited ICl(Ca) elicited by bath application of Ca2+ to oocytes permeabilized using the Ca2+ ionophore A23187, demonstrating that the inhibition of ICl(Ca) is due to a direct interaction with the Cl- channel, rather than by interference with Ca2+ entry through voltage-dependent Ca2+ channels. In addition to their use in the elimination of ICl(Ca) as a possible source of artifact when Xenopus oocytes are used as an expression system for exogenous ion channels and receptors, it is expected that these two compounds will find use as potent anion channel blockers.