Centre-surround inhibition--the suppression of activity of neighbouring cells by a central group of neurons--is a fundamental mechanism that increases contrast in patterned sensory processing. The initial stage of neural processing in olfaction occurs in olfactory bulb glomeruli, but evidence for functional interactions between glomeruli is fragmentary. Here we show that the so-called 'short axon' cells, contrary to their name, send interglomerular axons over long distances to form excitatory synapses with inhibitory periglomerular neurons up to 20-30 glomeruli away. Interglomerular excitation of these periglomerular cells potently inhibits mitral cells and forms an on-centre, off-surround circuit. This interglomerular centre-surround inhibitory network, along with the well-established mitral-granule-mitral inhibitory circuit, forms a serial, two-stage inhibitory circuit that could enhance spatiotemporal responses to odours.

We investigated the cellular mechanism underlying presynaptic regulation of olfactory receptor neuron (ORN) input to the mouse olfactory bulb using optical-imaging techniques that selectively report activity in the ORN presynaptic terminal. First, we loaded ORNs with calcium-sensitive dye and imaged stimulus-evoked calcium influx in a slice preparation. Single olfactory nerve shocks evoked rapid fluorescence increases that were largely blocked by the N-type calcium channel blocker omega-conotoxin GVIA. Paired shocks revealed a long-lasting suppression of calcium influx with approximately 40% suppression at 400-ms interstimulus intervals and a recovery time constant of approximately 450 ms. Blocking activation of postsynaptic olfactory bulb neurons with APV/CNQX reduced this suppression. The GABA(B) receptor agonist baclofen inhibited calcium influx, whereas GABA(B) antagonists reduced paired-pulse suppression without affecting the response to the conditioning pulse. We also imaged transmitter release directly using a mouse line that expresses synaptopHluorin selectively in ORNs. We found that the relationship between calcium influx and transmitter release was superlinear and that paired-pulse suppression of transmitter release was reduced, but not eliminated, by APV/CNQX and GABA(B) antagonists. These results demonstrate that primary olfactory input to the CNS can be presynaptically regulated by GABAergic interneurons and show that one major intracellular pathway for this regulation is via the suppression of calcium influx through N-type calcium channels in the presynaptic terminal. This mechanism is unique among primary sensory afferents.

Whole-cell patch-clamp recordings were used to investigate the electrophysiological properties of mitral cells in rat main olfactory bulb brain slice preparations. The majority of mitral cells are bistable. These cells spontaneously alternate between two membrane potentials, separated by ϳ10 mV: a relatively depolarized potential (upstate), which is perithreshold for spike generation, and a relatively hyperpolarized potential (downstate), in which spikes do not occur. Bistability occurs spontaneously in the absence of ionotropic excitatory or inhibitory synaptic inputs. Bistability is voltage dependent; transition from the downstate to the upstate is a regenerative event activated by brief depolarization. A brief hyperpolarization can switch the membrane potential from the upstate to the downstate. In response to olfactory nerve (ON) stimulation, mitral cells in the upstate are more likely to fire an action potential than are those in the downstate. ON stimulation can switch the membrane potential from the downstate to the upstate, producing a prolonged and amplified depolarization in response to a brief synaptic input. We conclude that bistability is an intrinsic property of mitral cells that is a major determinant of their responses to ON input. Key words: mitral cell; bistability; main olfactory bulb; plateau potential; upstate; downstate; response to olfactory nerveThe initial site of olfactory processing in vertebrate species is the main olfactory bulb (MOB). The MOB is a phylogenetically conserved cortical structure, with well defined architecture (Ramon y Cajal, 1894). The principal neurons of the MOB, the mitral cells, are located in a single lamina, the mitral cell layer (MCL); their dendrites span the MOB. The olfactory nerve (ON) synapses with mitral cell apical dendrites in the olfactory glomeruli, where interactions with juxtaglomerular interneurons occur. Mitral cell lateral dendrites interact with inhibitory granule cells, within the external plexiform layer (EPL). Although the morphology, projections, and synaptic interactions of mitral cells have been described , only recently have in vitro mammalian MOB preparations allowed detailed study of mitral cell electrophysiology.Recent investigations using rodent brain slices have elucidated neurotransmission between the ON and mitral cells Aroniadou-Anderjaska et al., 1997;Ciombor et al., 1999) and between mitral cell lateral dendrites and granule cells (Isaacson and Strowbridge, 1998;Schoppa et al., 1998; AroniadouAnderjaska et al., 1999a;Chen et al., 2000). Other studies have provided evidence of excitatory interactions among mitral cell lateral dendrites (Aroniadou-Anderjaska et al., 1999a,b;Isaacson, 1999;Friedman and Strowbridge, 2000) and intraglomerular excitatory interactions among mitral cell apical dendrites (Carlson et al., 2000).Neurons express a wide variety of electroresponsive properties (Llinas, 1988). Specific intrinsic membrane properties can endow individual neurons with multiple firing thresholds, levels of excitability, and modes ...

Heinbockel, Thomas, Philip Heyward, Fraçois Conquet, and Matthew Ennis. Regulation of main olfactory bulb mitral cell excitability of metabotropic glutamate receptor mGluR1. J Neurophysiol 92: 3085-3096, 2004. First published June 22, 2004 10.1152/ jn.00349.2004. In the rodent main olfactory bulb (MOB), mitral cells (MCs) express high levels of the group I metabotropic glutamate receptor (mGluR) subtype, mGluR1. The significance of this receptor in modulating MC excitability is unknown. We investigated the physiological role of mGluR1 in regulating MC activity in rat and mouse MOB slices. The selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), but not group II or III agonists, induced potent, dose-dependent, and reversible depolarization and increased firing of MCs. These effects persisted in the presence of blockers of fast synaptic transmission, indicating that they are due to direct activation of mGluRs on MCs. Voltage-clamp recordings showed that DHPG elicited a voltage-dependent inward current consisting of multiple components sensitive to potassium and calcium channel blockade and intracellular calcium chelation. MC excitatory responses to DHPG were absent in mGluR1 knockout mice but persisted in mGluR5 knockout mice. Broad-spectrum LY341495, MCPG, as well as preferential mGluR1 LY367385 antagonists blocked the excitatory effects of DHPG and also potently modulated MC spontaneous and olfactory nerve-evoked excitability. mGluR antagonists altered spontaneous membrane potential bistability, increasing the duration of the up and down states. mGluR antagonists also substantially attenuated MC responses to sensory input, decreasing the probability and increasing the latency of olfactory nerve-evoked spikes. These findings suggest that endogenous glutamate tonically modulates MC excitability and responsiveness to olfactory nerve input, and hence the operation of the MOB circuitry, via activation of mGluR1.

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 microM), isoproterenol (Isop, 10 microM), and clonidine (3 microM), were used to test for the functional presence of alpha1-, beta-, and alpha2-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 microM) induced an inward current (54 +/- 7 pA, n = 16) with an EC(50) of 4.7 microM. 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 microM), and blockers of excitatory and inhibitory fast synaptic transmission [gabazine 5 microM, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 microM, and (+/-)-2-amino-5-phosphonopentanoic acid (APV) 50 microM], the inward current induced by PE persisted (EC(50) = 9 microM), whereas that of Isop was absent. The effect of PE was also observed in the presence of the Ca(2+) channel blockers, cadmium (100 microM) and nickel (100 microM). The inward current caused by PE was blocked when the interior of the cell was perfused with the nonhydrolyzable GDP analogue, GDPbetaS, indicating that the alpha1 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 alpha1 receptors, an effect that may underlie, at least in part, increased mitral cell responses to weak ON input during locus coeruleus activation in vivo.

We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole-cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10nM), identifying it as a Na⁺ current. Slowly inactivating inward current, activated at potentials more positive than –30 mV, remained in Na⁺-free medium or in the presence of 1 µM TTX. This current was abolished by ionic Ca²⁺ channel blockade. In the presence of nifedi-pine about 70% of this high voltage-activated Ca²⁺ current was abolished, leaving a slowly inactivating component. No transient Ca²⁺ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 µM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K⁺ currents sensitive to membrane voltage and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were present. The resting membrane potential lay between –20 and –75 mV (mean = –43 ± 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at –20 to –35 mV) for up to 1 h. Apamin, the SK-type Ca²⁺-dependent K⁺ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca²⁺ channel blockade did not. Microfluorimetric detection of [Ca²⁺]_i showed that 10nM GnRH induced [Ca²⁺]i oscillations. We conclude that Ca²⁺ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca²⁺]_i fluctuations may activate the apamin-sensitive, Ca²⁺-dependent SK-type K⁺ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na⁺ current spikes may be crucial for activation of the nifedipine- and CGTX-sensitive high-voltage-activated Ca²⁺ channels.

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