Calcium signaling in mitral cell dendrites of olfactory bulbs of neonatal rats and mice during olfactory nerve Stimulation and beta-adrenoceptor activation

Abstract

Synapses formed by the olfactory nerve (ON) provide the source of excitatory synaptic input onto mitral cells (MC) in the olfactory bulb. These synapses, which relay odor-specific inputs, are confined to the distally tufted single primary dendrites of MCs, the first stage of central olfactory processing. beta-adrenergic modulation of electrical and chemical signaling at these synapses may be involved in early odor preference learning. To investigate this possibility, we combined electrophysiological recordings with calcium imaging in olfactory bulb slices prepared from neonatal rats and mice. Activation of ON-MC synapses induced postsynaptic potentials, which were associated with large postsynaptic calcium transients. Neither electrical nor calcium responses were affected by beta-adrenergic agonists or antagonist. Immunocytochemical analysis of MCs and their tufted dendrites revealed clear immunoreactivity with antibodies against alpha1A (Cav2.1, P/Q-type) and alpha1B (Cav2.2, N-type), but not against alpha1C (Cav1.2, L-type) or alpha1D (Cav1.3, L-type) calcium channel subunits. Moreover, nimodipine, a blocker of L-type calcium channels, had no effect on either electrical or calcium signaling at ON-MC synapses. In contrast to previous evidence, we concluded that in neonatal rats and mice (P5-P8), mitral cells do not express significant amounts of L-type calcium channels, the calcium channel type that is often targeted by beta-adrenergic modulation. The absence of beta-adrenergic modulation on either electrical or calcium signaling at ON-MC synapses of neonatal rats and mice excludes the involvement of this mechanism in early odor preference learning.

Figure 1

Morphology of a mitral cell (MC) in an olfactory bulb slice of a P6 mouse and positions of electrodes. The MC was filled through the patch-clamp electrode (also recording pipette, R) with 100 μM Oregon Green and fluorescence was imaged. Stimulation pipette (S) was placed in the olfactory nerve layer (ONL). (GL) Glomerular layer; (EPL) external plexiform layer; (MCL) mitral cell layer; (GCL) granule cell layer. (T) The tuft of the mitral cell apical dendrite. Scale bar, 50 μm.

Figure 2

Olfactory nerve (ON) stimulation-induced electrical and Ca²⁺ signaling. (A) Sub- and supra-threshold excitatory postsynaptic potentials (EPSPs) were evoked by single stimulation pulses (arrows) to the ON at varying intensities and recorded from the soma of MCs. (B) Time course of Ca²⁺ signals in a tufted dendritic terminal recorded simultaneously with electrical signals shown in A. (C) Map of increase in [Ca²⁺]_i measured at time points indicated in B.

Figure 3

Effect of NMDA receptor blockade on ON stimulation-induced glomerular Ca²⁺ signals. (A) Postsynaptic electrical (top traces) and Ca²⁺ signals (bottom traces) in control ACSF, in the presence of D-APV (50 μM), and after washout of D-APV. Arrows indicate times of ON stimulations. Note that D-APV reduced both Ca²⁺ signal and EPSPs. (B) Statistical analysis of experiments illustrated in A. Ca²⁺ signals and EPSPs were normalized to the responses obtained in control ACSF and expressed as the mean ± SEM (n = 7). Asterisks indicate statistical significant difference as compared with control (P < 0.005).

Figure 4

The effects of β-adrenoceptor agonists and antagonist on ON stimulation-induced glomerular responses in neonatal rats. (A) Effects of β-adrenoceptor agonists and antagonist on glomerular Ca²⁺ signaling. (B) Effects of β-adrenoceptor agonists and antagonist on EPSPs. Ca²⁺ signals of the tufted dendritic terminals and membrane potentials were recorded simultaneously. Note that application of β-adrenoceptor ligands exhibited no significant effect on either the Ca²⁺ signal or the EPSPs. Ca²⁺ signals and EPSPs were normalized to the responses obtained in control ACSF and expressed as the mean ± SEM. Isoproterenol (1-10 μM, n = 4), standard β-adrenoceptor agonist; xamoterol (1 μM, n = 2), β₁-adrenoceptor partial agonist; betaxolol (1 μM, n = 4), selective β₁-adrenoceptor antagonist.

Figure 5

The effects of isoproterenol on ON stimulation induced glomerular responses in neonatal mice. (A) Effects of isoproterenol (1-10 μM) on glomerular Ca²⁺ signals. Responses to sub- and supra-threshold stimulations (i.e., with and without action potential) are shown separately. (B) Effects of isoproterenol on EPSPs with and without action potentials. Ca²⁺ signals of the glomeruli and membrane potentials were recorded simultaneously. Note that isoproterenol exhibited no significant effect on either the Ca²⁺ signal or the EPSPs. Ca²⁺ signals and EPSPs were normalized to the responses obtained in control ACSF and expressed as the mean ± SEM (n = 14 and n = 5 for sub- and supra-threshold stimulations, respectively).

Figure 6

Immunocytochemical locations of VGCC subunits in neonatal rat (P7, n = 6) olfactory bulbs. Panels 1 through 4 indicate the immunoreactivity with antibodies against α1A (P/Q type; 6A), α1B (N-type; 6B), α1C (L-type; 6C) and α1D (L-type; 6D) distinctively. α1A subunit was located mainly in the glomeruli (A1, 20×; A2, 60×) and mitral cell soma (A3, 60×). α1B subunit antibody stained strongly all major neurons including mitral cells, granule cells (B1, 20×; B3, 60×), periglomerular cells (B1; B2, 60×) and also glomeruli (B1, B2). On the contrary, α1C antibody stained heavily in the EPL and IPL (C1, 20×; C3, 60×). However, mitral cell and granule cell soma (C1 and C3), and glomeruli (C1 and C2, 60×) exhibited no immunoreactivity. α1D antibody did not produce any specific staining in the OB (D1, 20×; D2 and D3, 60×). Arrows indicate MCs. Bars, 50μm.

Figure 7

The effects of nimodipine, a blocker of L-type voltage-gated calcium channels, on synaptic transmission at ON-MC synapses. (A) Electrical and calcium signaling at ON-MC synapses before, during, and after application of nimodipine (10 μM) to the bath solution. (B) summary data (mean ± SEM, n = 4) obtained as illustrated in A. Note that nimodipine exhibited no significant effect on either the Ca²⁺ signal or the EPSPs. Ca²⁺ signals, and EPSPs were normalized to the responses obtained in control ACSF and expressed as the mean ± SEM. (C) Effect of nimodipine (10 μM) on the field EPSPs evoked by ON stimulation and recorded in glomeruli. (Inset) Field EPSPs before (dotted line) and during (solid line) application of nimodipine. Horizontal lines demark the amplitude of the field EPSP. (D) Summary data (mean ± SEM, n = 5) obtained as illustrated in C. NPD, nimodipine.