Adrenergic receptor-mediated disinhibition of mitral cells triggers long-term enhancement of synchronized oscillations in the olfactory bulb

Abstract

Norepinephrine (NE) is widely implicated in various forms of associative olfactory learning in rodents, including early learning preference in neonates. Here we used patch-clamp recordings in rat olfactory bulb slices to assess cellular actions of NE, examining both acute, short-term effects of NE as well as the relationship between these acute effects and long-term cellular changes that could underlie learning. Our focus for long-term effects was on synchronized gamma frequency (30-70 Hz) oscillations, shown in prior studies to be enhanced for up to an hour after brief exposure of a bulb slice to NE and neuronal stimulation. In terms of acute effects, we found that a dominant action of NE was to reduce inhibitory GABAergic transmission from granule cells (GCs) to output mitral cells (MCs). This disinhibition was also induced by clonidine, an agonist specific for alpha(2) adrenergic receptors (ARs). Acute NE-induced disinhibition of MCs appeared to be linked to long-term enhancement of gamma oscillations, based, first, on the fact that clonidine, but not agonists specific for other AR subtypes, mimicked NE's long-term actions. In addition, the alpha(2) AR-specific antagonist yohimbine blocked the long-term enhancement of the oscillations due to NE. Last, brief exposure of the slice to the GABA(A) receptor antagonist gabazine, to block inhibitory synapses directly, also induced the long-term changes. Acute disinhibition is a plausible permissive effect of NE leading to olfactory learning, because, when combined with exposure to a specific odor, it should lead to neuron-specific increases in intracellular calcium of the type generally associated with long-term synaptic modifications.

Fig. 1.

Norepinephrine (NE) acutely decreases GABAergic transmission at granule (GC) to mitral cell (MC) synapses. A: an example experiment in which GC-to-MC GABAergic transmission was isolated by recording inhibitory postsynaptic currents (IPSCs) in a MC in response to electrical stimulation in the granule cell layer (GCL). Glutamate receptor antagonists were added to block MC-to-GC transmission. The 4 traces (averages of 10 responses) show that NE caused a reversible decrease in the amplitude of the gabazine (GBZ)-sensitive IPSC. A high-chloride-containing patch-pipette solution was used, resulting in inward-going IPSC events at the –70 mV holding potential. Stimulus artifacts were deleted in the displayed traces. B: the time plot for the experiment in A. C: a reversible reduction in evoked MC IPSCs was also observed in response to patterned olfactory nerve (ON) stimulation at the theta frequency (see cartoon trace of stimulus pattern at bottom). D: time plot for the experiment in C. IPSC activity was quantified from the current variance.

Fig. 2.

NE-effects on miniature IPSCs (mIPSCs) in MCs from rats of different ages. A: an example recording of mIPSCs in a bulb slice taken from a rat pup in our standard age group (P9-13). Raw traces (left) and averages of detected mIPSCs (right; averages of 36 events) are shown under control conditions and in the presence of NE. The amplitudes of the averaged mIPSCs were not significantly altered by NE. Recordings were done in the presence of glutamate receptor blockers (NBQX and d-AP5) and TTX (1 μM). B: time plot of mIPSC frequency for the experiment in A, showing that NE had no significant effect. Note that the GABA_A receptor blocker bicuculline methiodide (BMI; 10 μM) blocked the mIPSCs. C and D: NE caused an increase in the mIPSC frequency in a MC from an older P19 rat pup. The effect can be clearly observed in the raw example data traces (C, left) as well as in the time plot of frequency measurements in D. In this experiment, there was a ∼2-fold increase in the size of the detected mIPSC after NE application (C, right), though the effect likely reflected an inability to detect small mIPSC events under the conditions of very high mIPSC frequency in NE rather than an actual effect of NE on mIPSC amplitude.

Fig. 3.

α₂ adrenergic receptors (ARs) mediate disinhibition at GC-to-MC synapses. A: the α₂ AR-specific agonist clonidine, like NE, caused a reversible decrease in IPSCs in MCs evoked by GCL stimulation. Each trace from the displayed experiment reflects an average of 10 responses. B: time plot for the experiment in A. C and D: isoproterenol (Isop), an agonist specific for β ARs, had no effect on IPSCs evoked by GCL stimulation. Current records (C) and time plot (D) are shown. E: summary of effects of various AR-specific agonists on MC IPSCs evoked by GCL stimulation. Significant decreases in IPSCs were caused by NE and clonidine but not agonists specific for β ARs (Isop) or α₁ ARs (phenylephrine, PE). F: the NE-induced reduction in the IPSC evoked by GCL stimulation was mainly eliminated when NE was co-applied with the α₂ AR-specific antagonist yohimbine (20 μM), further supporting a role for α₂ ARs. Each trace reflects an average of 10 responses. G and H: recordings of mIPSCs in the presence of TTX, showing that clonidine had no effect on the frequency. Current records (G) and time plot of mIPSC frequency (H) are shown.

Fig. 4.

NE effects on glutamatergic signaling onto GCs. A and B: NE had no effect on excitatory postsynaptic currents (EPSCs) recorded in GCs evoked by electrical stimulation in the external plexiform layer (EPL). These EPSCs, with moderate ∼10 ms decay kinetics, reflect dendrodendritic transmission from MC lateral dendrites (see arrow and “+” in the cartoon in A). Recordings were done in the presence of the GABA_A receptor blocker BMI (10 μM). C and D: NE reversibly reduced EPSCs in GCs evoked by stimulation in the granule cell layer. These EPSCs, with rapid decay kinetics, appear to at least partly reflect glutamatergic centrifugal inputs from the piriform cortex (Balu et al. 2007). Each trace in A and C reflects an average of 10 responses.

Fig. 5.

NE plus N-methyl-d-aspartate (NMDA) as a conditioning stimulus leads to a long-term increase in synchronized oscillations. A: LFPs recorded in the EPL, showing slow oscillatory responses evoked by bath-applied NMDA (12.5 μM). Data were filtered at 0.5–100 Hz. B: protocol used to examine effects of various conditioning stimuli on gamma oscillations in LFP signals in the EPL. Following a control period, a conditioning stimulus was applied for 10 min, which was then followed by a test period initiated on washout of the conditioning drugs. In the control and test periods, test stimuli were applied that consisted of patterned stimulation of the ON at the theta frequency. C: LFP responses to ON stimulation before and after application of a conditioning stimulus that included NE (20 μM) and NMDA (12.5 μM). Note the enhancement in the oscillatory signal, seen also in the enlarged insets at right. Data were filtered at 10–100 Hz. D: power spectra (between 10 and 100 Hz) derived from the same experimental recordings as in C. Note that the NE-plus-NMDA conditioning stimulus increased the power across the 10–100 Hz range with a particularly strong effect around 40–50 Hz. The spectrum for the postconditioning stimulus-condition reflects recordings made 50–60 min following washout of NE-plus-NMDA. Only a limited frequency range in the power spectra, down to 10 Hz, is shown, because LFP signals had to be analyzed in data segments constrained by the patterned ON stimulation protocol (see C and methods). No data point is shown at 60 Hz to eliminate possible contamination from line-noise. E: time plot for the experiment in C, showing changes in the integrated power of gamma oscillations between 30–70 Hz following removal of the conditioning stimulus. F: summary of the effects of different conditioning stimuli on gamma oscillations (NE-plus-NMDA, NE alone, or NMDA alone). All test measurements for the summary were made 30–60 min following washout of the conditioning drugs. Also shown are the results when no conditioning stimulus was applied. These data reflect the magnitude of the oscillations recorded 50–60 min after the start of the recording as compared with the first 10 min.

Fig. 6.

The α₂ AR-specific agonist clonidine induces a long-term increase in gamma oscillations. A: consecutive traces of LFP recordings of responses to ON stimulation made under control conditions and ∼60 min following washout of a conditioning stimulus comprised of clonidine (5 μM) and NMDA (12.5 μM). Note the large increase in the oscillatory signal following the conditioning stimulus, also shown in the boxed insets at right. Data were filtered at 10–100 Hz. B: power spectra derived from the same experimental recordings as in A, showing that the clonidine-plus-NMDA conditioning stimulus increased the oscillatory power across the 10–100 Hz range. C: time plot of the experiment in A, showing a long-term increase in the integrated power of gamma oscillations between 30 and 70 Hz due to a clonidine-plus-NMDA conditioning stimulus. D: summary of the effects of various conditioning stimuli on the integrated power of the LFP oscillations between 30 and 70 Hz. Results are shown for drug combinations that included NMDA plus agonists specific for different AR subtypes (Clon, clonidine; Isop, isoproteronol; PE, phenylephrine). E: results from another experiment showing that the NE plus NMDA conditioning stimulus failed to enhance the LFP oscillations (integrated power between 30 and 70 Hz) when the drugs were co-applied with the α₂ AR-specific antagonist yohimbine (20 μM).

Fig. 7.

Direct pharmacological block of GABA_A receptors leads to long-term enhancement of gamma oscillations. A: consecutive traces of LFP recordings of responses to ON stimulation, made under control conditions and ∼30 min following washout of a conditioning stimulus comprised of gabazine (GBZ; 5 μM) and NMDA (12.5 μM). Note the large increase in the oscillatory signal following removal of the conditioning stimulus, also shown in the boxed insets at right. Data were filtered at 10–100 Hz. B: time plot of the experiment in A, showing a long-term increase in the integrated power of gamma oscillations between 30 and 70 Hz due to the GBZ-plus-NMDA conditioning stimulus. Note also that, just following the conditioning stimulus, there was a moderate decrease in oscillations that preceded the enhancement (66% decrease during minutes 30–32 vs. control period). This decrease was presumably due to residual GBZ prior to washout. In this experiment, the first 50 ms of data following each ON stimulus burst was ignored in the analysis to avoid a lower-frequency component of the response (see traces in A).