Age-dependent adrenergic actions in the main olfactory bulb that could underlie an olfactory-sensitive period

Abstract

Many sensory systems are endowed with mechanisms of neural plasticity that are restricted to a sensitive period in the young developing animal. In this study, we performed experiments in slices of the main olfactory bulb (OB) from rats to examine possible age-dependent cellular mechanisms of plasticity in the olfactory system. We focused on the neurotransmitter norepinephrine (NE), shown to be important in different forms of olfactory learning, examining whether two specific cellular effects of NE previously observed in rats less than P14 extended to older animals. These included an acute reduction in GABAergic synaptic transmission from granule cells (GCs) onto output mitral cells (MCs) and an enhancement in gamma frequency (30–70 Hz) oscillations that persists long after removal of NE. We found that NE failed to reduce GC-to-MC transmission or enhance gamma oscillations in older rats at P18–23. The loss of NE actions on both phenomena appeared to reflect an age-dependent loss of function of α₂-adrenergic receptors. In addition, we found that NE induced an age-dependent enhancement of transient excitation in MCs, providing a mechanism to link the acute decrease in GC-to-MC inhibition to the long-term increase in gamma oscillations through increases in intracellular calcium. The age-dependent cellular mechanisms that we describe could underlie an olfactory-sensitive period in newborn rodents.

the neural circuits of many sensory systems are shaped by experience early in an animal's postnatal life (Hensch 2004; Maffei and Turrigiano 2008), when a well-defined set of molecular and cellular events alter neural growth, synaptic connections, and expression of transmitter receptors. For olfaction, there is strong behavioral evidence for a number of neural plasticity mechanisms with sensitive periods (Brennan and Keverne 1997; Shingo et al. 2003), including some that are restricted to very young animals. For example, newborn rats display a form of associative learning in which an animal develops a preference for odors applied with a positively reinforcing stimulus (Moriceau and Sullivan 2004; Sullivanet al. 1989; Woo and Leon 1987). This odor preference learning, which appears to subside in <2 wk after birth, has been associated with changes in neural activity in the first olfactory processing center, the olfactory bulb (OB; Coopersmith and Leon 1984; Johnson and Leon 1996; Wilson et al. 1987). Another form of behavioral plasticity restricted to the first 3 days of life occurs in newborn rabbits, in which pups associate a novel odor with suckling (Hudson 1993). The cellular events that could govern the timing of such age-dependent neural plasticity mechanisms are, however, not well understood.

In searching for candidate cellular mechanisms, a key component is likely to be the neurotransmitter norepinephrine (NE) released in the OB from inputs from the locus coeruleus. NE is critical for many forms of olfactory learning at the behavioral level (Brennan et al. 1995; Doucette et al. 2007; Shea et al. 2008; Wilson and Sullivan 1994) and has been shown, in cellular studies, to have a number of effects on the OB in young animals. In OB slices from P9–13 rats, NE through α₂-adrenergic receptor (AR) activation can markedly increase gamma frequency (30–70 Hz) synchronized oscillations recorded in the external plexiform layer (EPL) that can persist for at least an hour after removal of NE (Gire and Schoppa 2008; Pandipati et al. 2010). These gamma oscillations reflect dendrodendritic synaptic interactions between GABAergic granule cells (GCs) and output mitral cells (MCs) (Rall and Shepherd 1968), and their long-lasting enhancement could modify integration of MC inputs by downstream olfactory cortical structures (Mori et al. 1999). NE through β-AR activation can also elicit long-lasting increases in MC excitatory responses following olfactory nerve (ON) stimulation in very young rats (Lethbridge et al. 2012; Yuan 2009; Yuan et al. 2000). In terms of acute effects, NE can reduce inhibitory GC-to-MC transmission in rodents less than P14 (Pandipati et al. 2010; Wilson and Leon 1988). One clue that at least the acute effect of NE on reducing GC-to-MC synaptic transmission could be specific to very young rodents comes from studies in somewhat older P14–30 rats done by Nai and coworkers (2009). They found that NE can increase the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) when applied at a concentration that decreased GC-to-MC inhibitory transmission in P9–13 rats (Pandipati et al. 2010). Comparisons across studies are, however, complicated by the different methods used to isolate GC-to-MC transmission (sIPSCs vs. IPSCs evoked by electrical stimulation).

Here we tested the hypothesis that the specific action of NE in OB is dependent on the age of the animal, with patch-clamp recordings done in rat OB slices. We compared the effects of NE on rats around postnatal 3 wk to younger rats at P9–13. Evidence is provided that the long-term enhancement in gamma oscillations and acute disinhibition is restricted to younger rats, with the change apparently reflecting a shift in function of α₂-ARs. In addition, disinhibition drives an increase in MC depolarization that is restricted to young animals. Our results provide a plausible mechanism that could account for a sensitive period of NE-mediated neural plasticity in the olfactory system.

MATERIALS AND METHODS

All experiments were approved by the Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus.

Electrophysiological recordings in OB slices.

The experiments described here were all performed in mixed-sex wild-type Sprague-Dawley rat pups ranging in age from P9 to P23. Recordings were performed in 300- to 330-μm-thick horizontal OB slices that were prepared as previously described (Schoppa et al. 1998), except that a high sucrose-containing solution was used during slicing and slice storage. This solution contained (in mM) 72 sucrose, 83 NaCl, 26 sodium bicarbonate, 10 glucose, 1.25 sodium phosphate monobasic, 3.5 KCl, 3 MgCl₂, and 0.5 CaCl_2. Slices were viewed under differential interference contrast optics at ×40 magnification (Axioskop; Carl Zeiss, Thornwood, NY). All experiments were done at 31–33°C.

For all recordings, the base extracellular solution contained (in mM) 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 25 glucose, 3 KCl, 2 CaCl₂, and 1 MgCl₂ (pH 7.3) and was oxygenated (95% O₂, 5% CO₂). In recordings of gamma local field potential (LFP) oscillations, the bath solution included 100 μM sulpiride, a dopamine D₂ receptor antagonist, in order to block possible inhibitory effects of NE on presynaptic ON terminals through D₂ receptors (Ennis et al. 2001; Hayar et al. 2001). LFPs were recorded with glass micropipettes filled with extracellular solution (resistance 1–5 MΩ) placed in the EPL, within 50 μm of the MC layer. For whole cell recordings of IPSCs in MCs, the intracellular pipette solution contained (in mM) 125 KCl, 2 MgCl₂, 0.025 CaCl₂, 1 EGTA, 2 NaATP, 0.5 NaGTP, and 10 HEPES (pH 7.3 with KOH). For recordings of NMDA-evoked slow oscillations in MCs, a pipette solution was used in which the intracellular KCl was replaced with equimolar K-gluconate. The holding potential for all voltage-clamp recordings was −70 mV. All whole cell and extracellular recordings were made with a Multi-Clamp 700B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) and were low-pass filtered at 0.25–5 kHz with an eight-pole Bessel filter and digitized at 0.5–10 kHz. Data were acquired with Axograph software (Molecular Devices) on a Macintosh Pro.

For electrical stimulation of the ON, a glass patch pipette (0.5–2 MΩ; broken-tip diameter of 2–4 μm) containing the extracellular solution was placed in the ON layer. A theta frequency pattern was applied with a biphasic stimulus isolation unit (A365 Stimulus Isolator; World Precision Instruments, Sarasota, FL). The pattern consisted of five short bursts (3 pulses at 100 Hz) separated by 250 ms (4 Hz overall). Each stimulus pattern was applied every 30 s. For whole cell recordings of IPSCs, stimulation pipettes were placed in the GC layer. Stimuli were applied every 20 s.

In all experiments, test drugs were bath-applied through a constant-flow perfusion system.

Data analysis.

Analysis was done with AxoGraph X (AxoGraph Scientific, Sydney, Australia), as well as custom-written software in MATLAB (release 7.1, The MathWorks, Natick, MA). Except where noted, statistical significance was determined via Student's t-test. Data values are reported as means ± SE.

For the analysis of the LFP oscillations evoked by theta frequency stimulation, traces underwent supplementary filtering off-line (8-pole band-pass Butterworth filter at 10–100 Hz) prior to further analysis. To detect oscillatory activity, a fast Fourier transform (FFT) was performed on filtered LFP recordings; data were analyzed in 200-ms sections following the end of each electrical stimulus burst. The resulting power spectra were integrated from 30 Hz to 70 Hz, and this value was used as a measure of the magnitude of gamma frequency oscillatory activity. In some recordings, an additional small peak at 60 Hz reflecting line noise appeared in the spectra; for these experiments, spectra were integrated between 30 and 55 Hz.

For the analysis of acute effects of NE and clonidine on evoked MC IPSCs, comparisons between control and drug conditions were made by comparing the average of 5–10 traces under each condition. sIPSCs were detected with peak detection software in AxoGraph. In the analysis of the NMDA-evoked slow oscillations (see Fig. 5), traces for analysis did not require additional filtering prior to construction of low-frequency power spectra, owing to the large magnitude of the slow oscillations.

Fig. 5.

ARs mediate age-dependent enhancement of MC excitation. A: expanded section of the NMDA-evoked current response in a MC from a P10 rat, taken from data trace in Fig. 1E. Note the period of higher current variance during the peak of the inward current phase of the slow oscillation. This reflects, at least partially, inhibitory synaptic activity. B, top: traces of MC current recorded in the presence of NMDA (holding potential = −70 mV), showing that blockade of inhibition with GBZ (5 μM) enhanced the slow oscillations. Bottom: low-frequency power spectra from the same experiment, showing the GBZ-induced enhancement and slowing of the oscillations. Data were recorded in an OB slice from a P11 rat. C: blockade of α₂-ARs with clonidine (Clon; 5 μM) enhances the NMDA-evoked slow oscillation in a MC in a young P9 rat (left) but not in an older P20 rat (right). Raw data traces are shown at top and power spectra at bottom. D: summary of the effects of GBZ and clonidine on NMDA-evoked slow oscillations in MCs. Histogram at left shows an increase in the slow oscillation power (integrated between 0.1 and 1 Hz) caused by both GBZ and clonidine in young rats at P9–13 and the lack of an effect of clonidine in P18–23 rats. Histogram at right summarizes GBZ/clonidine actions on the kinetics of the slow oscillations, plotted as % change in the frequency at which the power spectra peaked. *P < 0.05.

RESULTS

NE-mediated long-term enhancement of gamma oscillations is age dependent.

Our first objective was to evaluate whether the pronounced long-term increase in gamma frequency oscillations that NE causes in P9–13 rats (Gire and Schoppa 2008; Pandipati et al. 2010) extended to older animals. For this analysis, we recorded LFPs by placement of a patch electrode in the EPL of OB slices and evoked gamma LFP oscillations with electrical theta-burst stimulation of the ON (10–500 μA; Fig. 1A). We used a dual-component conditioning stimulus that included bath-applied NE and a moderate concentration of NMDA (12.5 μM), designed to mimic the rise in NE and neural activation in OB that could drive AR-mediated behavioral modifications (Wilson and Sullivan 1994). Our prior studies of gamma oscillations done in young P9–13 animals used both NMDA (Pandipati et al. 2010) and electrical stimulation of ON (Gire and Schoppa 2008) as components of the conditioning stimulus to induce neural activation, but NMDA caused more consistent long-term increases in the gamma oscillations. We attribute the higher efficacy of NMDA to its ability to cause spatially broader activation of the OB. NMDA or electrical stimulation of ON can cause slow (0.5–2 Hz) oscillatory depolarizations across glomeruli (Schoppa and Westbrook 2001), yet NMDA applied to the entire slice presumably causes many more glomeruli to engage in the slow oscillations. As for the NE component of the conditioning stimulus, we used a 20 μM concentration. NE effects on OB circuit properties can vary with concentration (Nai et al. 2009), but we wanted to focus on one NE concentration shown previously to cause robust enhancement of gamma oscillations in P9–13 rats.

Fig. 1.

Norepinephrine (NE) fails to induce long-term enhancement of gamma frequency oscillations in P18–23 rats. A: recordings of local field potentials (LFPs) in the external plexiform layer (EPL) in an olfactory bulb (OB) slice from a P21 rat. Olfactory nerve (ON) stimulation (300 μA) applied as a theta frequency pattern (see bottom trace) elicited high-frequency oscillations that failed to be enhanced by a conditioning stimulus including NE (20 μM) and NMDA (12.5 μM). Three consecutively recorded LFP traces are shown for each condition. Boxes at right show expanded portions of the traces. Recordings made after the conditioning stimulus were done 30 min after washout of NE and NMDA. Displayed LFP traces were filtered at 10–100 Hz. Cartoon at left shows OB layers, including the ON layer, glomerular layer (GL), EPL, mitral cell (MC) layer (MCL), and granule cell (GC) layer (GCL). B: expanded portions of an LFP recording from a P12 rat, showing enhancement of the oscillations after washout of NE and NMDA. C: power spectra (between 10 and 100 Hz; top) and time plots of integrated power measurements (between 30 and 55 Hz; bottom) derived from the experiments shown in A (P21 rat) and B (P12 rat). Bars in the time plots denote the conditioning period, when NE and NMDA were applied. No ON test stimulus was applied during the conditioning period. Power spectra represent averages obtained during a 10-min control (Con) period just prior to the conditioning stimulus and during the last 10 min of each experiment, after the conditioning stimulus was removed. D: averaged power spectra obtained from rats at P18–23 (top; n = 10) and P9–13 (bottom; n = 8), showing that long-term enhancement of the gamma oscillations was specific to younger animals. To construct the plots, all data points for each experiment were normalized to the peak value of the power spectra measured under control conditions. Data at 60 Hz were excluded from the analysis, as some recordings included 60-Hz line noise. E: whole cell patch recording of MC current (holding potential = −70 mV) made in the presence of NMDA (12.5 μM). NMDA was similarly effective in evoking slow ∼0.5-Hz oscillations in P20 and P10 rats, indicating that the failure of the conditioning stimulus that included NE + NMDA to elicit long-term effects on gamma oscillations in older rats was not due to lack of action of NMDA. Area inside dashed box in P10 trace is shown in expanded view in Fig. 5A.

In experiments in OB slices from older rats at P18–23, we found that the conditioning stimulus that included NE and NMDA in fact failed to enhance gamma oscillations following washout of the drugs (Fig. 1, B–D; 24 ± 13% decrease in integrated power between 30 and 70 Hz during last 10 min of recordings vs. 10-min control period just prior to conditioning stimulus, n = 10, P = 0.09). Separating experiments by the magnitude of the drug effect, we found that NE plus NMDA caused a greater than twofold increase in gamma power in 5 of 8 recordings in P9–13 rats (Pandipati et al. 2010), but a greater than twofold increase was observed in none of 10 recordings in P18–23 rats. Similar age-dependent effects on gamma oscillations were observed if the α₂-AR-specific agonist clonidine (5 μM) replaced NE in the conditioning stimulus. Clonidine plus NMDA caused a greater than twofold increase in the gamma power in 8 of 23 recordings in P9–13 rats (Pandipati et al. 2010) but in only 1 of 14 recordings in P18–23 rats. In the one experiment in older rats in which there was a power increase, the increase was very large (11-fold). In statistical tests, there was no significant increase in gamma power in P18–23 rats regardless of whether that one experiment was included in the analysis (89 ± 67% increase, n = 14; P = 0.44 with Kolmogorov-Smirnov test) or not (18 ± 10% increase, n = 13; P = 0.09 with t-test).

One possible explanation for the general failure of the conditioning stimuli to enhance gamma oscillations in P18–23 rats was poor slice health. However, the resting membrane potentials in MCs were the same in slices from rats at P18–23 (−53.8 ± 1.2 mV, n = 25) versus P9–13 (−50.2 ± 1.4 mV, n = 25; P = 0.059), and sIPSC frequencies in MCs in P18–23 rats (2.6 ± 0.4 Hz, n = 13) were higher than in P9–13 rats (0.74 ± 0.09 Hz, n = 10; P = 0.0007), which suggests that GCs were healthy. In addition, we found in voltage-clamp recordings from MCs that NMDA still evoked pronounced slow oscillations in MCs [Fig. 1E; peak-to-trough amplitude = 31 ± 5 pA at P18–23 (n = 7) vs. 25 ± 2 pA at P9–13 (n = 7); P = 0.29]. Hence OB slices from older animals have active networks capable of responding to the NMDA component of the conditioning stimuli. The lack of effect of the conditioning stimulus on gamma oscillations in P18–23 rats also was not a result of a frequency shift in the fast oscillations, which could have altered the results of our analysis [power spectra peaks in P18–23 vs. P9–13 rats, respectively, were 38 ± 2 Hz (n = 10) and 41 ± 2 Hz (n = 10); P = 0.41]. Furthermore, on the basis of experiments described below, we can exclude the possibility that the absent effect of NE on gamma oscillations in older animals was due to poor drug penetration in older animal slices (see NE-induced increase in sIPSCs in Fig. 3A) or because gamma oscillations were already potentiated (see gabazine-induced increase in oscillations in Fig. 4). These results point to a changing acute action of NE during the conditioning stimulus.

Fig. 3.

AR-mediated actions on spontaneous IPSCs (sIPSCs). A: recording of sIPSCs in an OB slice from a P20 rat, showing that NE (20 μM) caused a rapid and large increase in the sIPSC frequency. Raw current traces are shown at left and a time plot of sIPSC frequency measurements at right. The NE-induced increase in sIPSC frequency, similar to that observed by Nai and coworkers (2009), differed from the noneffect of NE on evoked IPSCs in P18–23 rats (see Fig. 2A). B: the α₁-AR-specific agonist phenylephrine (PE; 50 μM) increased the frequency of sIPSCs in both P19 and P11 rats. Thus effects mediated by α₁-ARs do not appear to be age dependent. C: summary of NE and PE effects on the frequency of sIPSCs in P9–13 and P18–23 rats. *P < 0.05.

Fig. 4.

A conditioning stimulus that includes gabazine (GBZ) and NMDA causes long-term enhancement of gamma oscillations in older rats. A: LFP recordings made in the EPL in a P20 rat before and after a conditioning stimulus that contained the GABA_A receptor blocker GBZ (5 μM) and NMDA (12.5 μM). Note the marked increase in the oscillations recorded 30 min after washout of GBZ + NMDA. Boxes (right) show expanded traces. All displayed traces were filtered at 10–100 Hz. B: power spectra (10–100 Hz) derived from the experiment in A. Spectra reflect averages obtained during a 10-min control period just prior to the conditioning stimulus and during the last 10 min of the experiment, after washout of the conditioning stimulus. C: time plot of integrated power measurements (between 30 and 70 Hz) for the experiment shown in A. D: averaged power spectra obtained in P18–23 rats under control conditions and after exposure to and washout of GBZ and NMDA. All data points for each experiment were normalized to the peak value of the power spectra measured under control conditions.

Age-dependent loss of long-term effect of NE on gamma oscillations is due to loss of α₂-AR-mediated acute disinhibition.

Of the different acute effects that NE has been reported to have in OB slices, one that could drive a long-term enhancement of gamma oscillations that persists after NE has been removed is a reduction in GC-to-MC transmission mediated by α₂-ARs (Pandipati et al. 2010). Increased MC excitation resulting from such disinhibition could lead to rises in intracellular calcium implicated in many forms of long-lasting synaptic changes. To test whether there is a loss of α₂-AR-mediated reduction in the GC-to-MC inhibitory transmission in older animals, we measured IPSCs in MCs in response to electrical stimulation in the GC layer (100 μs; 50–200 μA) while including the glutamate receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 10 μM) and dl-2-amino-5-phosphonopentanoic acid (DL-APV, 50 μM) in the bath to eliminate MC-to-GC transmission. When a high chloride-containing intracellular pipette solution is used, such a protocol results in pronounced inward-going evoked IPSCs reflecting GC-to-MC transmission (Fig. 2A). In contrast to the ∼50% decrease in IPSC amplitude that NE causes in P9–13 rats (Pandipati et al. 2010), NE caused no effect in P18–23 rats (9 ± 16% decrease in IPSC amplitude, n = 15, P = 0.58; Fig. 2, A and C). We also observed a strong age dependence in the effect of the α₂-AR-specific agonist clonidine on evoked IPSCs. Clonidine (5 μM), which markedly reduces evoked IPSCs in P9–13 rats (Pandipati et al. 2010), had no effect in older animals (9 ± 15% decrease in IPSC amplitude, n = 6, P = 0.59; Fig. 2, B and C). Thus α₂-AR activation can no longer reduce GC-to-MC inhibitory transmission in P18–23 rats.

Fig. 2.

Age-dependent effects of α₂-adrenergic receptor (AR) activation on evoked GC-to-MC transmission. A: inhibitory postsynaptic currents (IPSCs) in MCs evoked by electrical stimulation in the GC layer at 2 ages, P20 (left) and P11 (right). While NE (20 μM) caused reversible reduction in the IPSC at P11, no effect was observed at P20. Time plots for the experiments are shown at bottom; horizontal bar indicates period of NE application. Each trace reflects average of 6–12 responses. B: effect of the α₂-AR-specific agonist clonidine (Clon; 5 μM) on the MC IPSC evoked by GC layer stimulation in P19 (left) and P11 (right) rats. As seen for NE in A, clonidine reduced the IPSC only in bulb slices taken from the younger animal. Average traces and time plots are shown at top and bottom, respectively. C: summary of NE and clonidine effects on evoked IPSCs in P9–13 and P18–23 rats. Data for P9–13 rats are taken from Pandipati et al. (2010). *P < 0.01.

We also measured sIPSCs as an assay of GC-to-MC transmission, since it has been reported that NE at the concentration that we used (20 μM) can increase the frequency of sIPSCs in P14–30 rats (Nai et al. 2009). An NE-induced increase in sIPSCs differs from the noneffect that we observed due to NE and clonidine on evoked IPSCs in P18–23 rats and the decrease in evoked IPSCs observed in P9–13 rats. We found that NE did increase the frequency of sIPSCs in our older rat group (by 86 ± 28%, n = 10, P = 0.01; Fig. 3, A and C; measured in the presence of NBQX and DL-APV), as seen by Nai and coworkers in P14–30 rats, although no effect was observed in P9–13 rats (12 ± 19% increase in sIPSC frequency, n = 10; P = 0.89). The difference in NE effects in older rats between evoked IPSCs (no effect) and sIPSCs (an increase) may reflect NE-induced depletion of GABA-containing vesicles or differences in the mechanisms of the inhibitory events. sIPSCs likely include a large number of miniature IPSCs (mIPSCs) that are independent of spikes in GCs, while evoked IPSCs are spike dependent. We also used recordings of sIPSCs to evaluate age-dependent actions of α₁-ARs, since sIPSC recordings have revealed large effects of α₁-AR activation on GC-to-MC transmission (Nai et al. 2009). The α₁-AR-specific agonist phenylephrine (50 μM) caused similar effects on sIPSC frequency at different ages [282 ± 131% increase at P9–13 (n = 11, P = 0.027), 299 ± 95% increase at P18–23 (n = 6, P = 0.0006); Fig. 3, B and C], suggesting that the function of α₁-ARs, unlike α₂-ARs, is not age dependent.

Did the loss of the acute effect of AR activation of GC-to-MC inhibitory transmission in older animals contribute to the loss of NE's long-term actions on gamma oscillations (Fig. 1)? To answer this question, we tested whether long-term changes in the oscillations could occur in P18–23 rats when inhibitory synapses were directly blocked by an antagonist of GABA_A receptors. If the loss in long-term changes due to NE were because of a loss of acute modulation of GC-to-MC transmission, a conditioning stimulus including a GABA_A receptor blocker and NMDA should still be effective in eliciting long-term changes in the gamma oscillations in older P18–23 rats. This is indeed what was found (Fig. 4, A–C). Gabazine (5 μM) plus NMDA increased the gamma oscillations in P18–23 rats by an amount (109 ± 43% increase in power, n = 8; P = 0.04) similar to that in P9–13 rats (105% average increase in power; Pandipati et al. 2010). Thus most of the mechanisms that drive long-term enhancement of gamma oscillations appear to be present in older animals, yet NE appears to no longer be able to engage them because of loss of its ability to modulate GC-to-MC transmission.

Impact of reduced GC-to-MC transmission on MC excitation.

Thus far, we have provided evidence that there is an age-dependent loss in the ability of a conditioning stimulus that includes NE and NMDA to drive long-term enhancement of gamma oscillations and that this is due to a loss of acute modulation of GC-to-MC transmission. One final question that we addressed pertained to our basic model for how reduced GC-to-MC transmission during the acute phase of a conditioning phase could be linked to long-term changes. As suggested above, reduced GC-to-MC transmission could increase MC excitation, leading to rises in intracellular calcium transients, but there is in fact some question as to whether a reduction in GC-to-MC transmission would effectively increase MC excitation. GC-to-MC transmission may be primarily involved in synchronizing spiking in MCs (Galán et al. 2006; Schoppa 2006) rather than affecting the degree of MC excitation (Gire and Schoppa 2009).

One clue to possible effects of reduced GC-to-MC transmission on MC excitation during the conditioning stimuli was provided by visual inspection of the current transients in MCs recorded in the presence of NMDA (see expanded trace in Fig. 5A). The excitatory phase of the evoked slow oscillations was typically associated with a barrage synaptic activity that was blocked by gabazine (5 μM; n = 12) and thus reflected at least in part inhibitory circuits. This GABAergic synaptic activity limited NMDA-evoked excitation of MCs, since gabazine greatly enhanced the slow oscillations [Fig. 5, B and D; 176 ± 53% increase in integrated power between 0.1 and 1 Hz (n = 6, P = 0.018); all data from P9–13 rats] and also prolonged them somewhat (13 ± 4% decrease in peak frequencies in power spectra, n = 6; P = 0.021). Gabazine did not alter the holding current in MCs measured in NMDA (−4 ± 10 pA shift, n = 6; P = 0.84). Thus GABAergic inhibition can impact MC excitation, specifically by altering the transient depolarization during the NMDA-evoked slow oscillation.

Similar to the effect of gabazine, we found that reducing inhibitory transmission onto MCs through AR activation also enhanced transient MC excitation in P9–13 rats. The α₂-AR-specific agonist clonidine (5 μM) increased the magnitude of the slow oscillations [107 ± 49% increase in integrated power between 0.1–1.0 Hz (n = 8, P = 0.01); Fig. 5, C and D], while also slowing their frequency (19 ± 7% decrease in peak frequency of power spectra, n = 8; P = 0.048). In addition, as predicted from the age-dependent loss in AR-mediated effects in GC-to-MC transmission, clonidine did not impact the slow oscillations in older animals at P18–23 [26 ± 24% increase in integrated power between 0.1 and 1.0 Hz (n = 5, P = 0.31); 14 ± 14% increase in peak frequency (n = 5)]. Thus α₂-AR-mediated reduction in inhibitory transmission provides a potent age-dependent mechanism to enhance MC excitation in rat OB.

DISCUSSION

In this study, we performed experiments in rat OB slices to test for age-dependent actions of NE on the cellular properties of the OB.

Age-dependent changes in cellular actions of NE in olfactory bulb.

We found that there was a strong age dependence to two previously reported actions of NE (Gire and Schoppa 2008; Pandipati et al. 2010). The first was in an NE-induced long-term increase in gamma frequency oscillations. In young animals, at P9–13, a conditioning stimulus that includes NE and neural excitation induces an increase in gamma oscillations that persists long after the conditioning stimulus has been removed, but no such effect was observed in older animals at P18–23. Second, NE caused an acute reduction in GABAergic transmission from GCs to MCs in young animals at P9–13 but not in older animals. At the level of specific AR subtypes, it appears that these age-dependent effects reflected a loss of action of α₂-ARs, since the α₂-AR-specific agonist clonidine mimicked the effect of NE on both acute and long-term effects in young but not older animals. The lack of an effect of α₂-AR activation after P13 could reflect reduced expression of α₂-ARs (Winzer-Serhan et al. 1997) or α₂-ARs having different physiological functions in older animals that were not studied here. This study focused on a concentration of NE (20 μM) that should activate multiple AR subtypes in the OB (Nai et al. 2009), yet α₂-ARs appeared to be most important in driving the age-dependent actions studied here. In recordings of sIPSCs, direct activation of α₁-ARs with phenylephrine caused a similar increase in sIPSC frequencies in young and older animals (Fig. 2C). As for β-ARs, we did not observe any effect of the β-AR-specific agonist isoproterenol on evoked IPSCs or gamma oscillations in our prior study done in P9–13 rats (Pandipati et al. 2010). Thus, while we did not explore age-dependent changes of β-ARs in this study, it is very unlikely that β-receptors contributed to the loss of an effect of NE as animals aged.

To what extent are the two primary age-dependent effects—the loss of α₂-AR-mediated reduction in GC-to-MC transmission and the loss of long-term changes in the gamma oscillations—causally linked to each other? Evidence supporting a link was provided by our experiments in which a conditioning stimulus that included NMDA and gabazine to block GABA_A receptors directly was able to induce a long-term increase in gamma oscillations in P18–23 rats (Fig. 4). This result suggests that most cellular mechanisms that lead to long-term enhancement of the gamma oscillations are present in older animals, except α₂-AR-mediated disinhibition. In addition, we demonstrated that α₂-AR activation can enhance the NMDA-evoked oscillatory depolarization in MCs in P9–13 but not P18–23 rats (Fig. 5), suggesting that α₂-AR-mediated disinhibition would enhance calcium transients that typically drive long-lasting synaptic changes. Our experiments do not exclude that there are differences in older animals other than loss of action of α₂-ARs that contribute to the loss of long-term effects of NE on gamma oscillations, for example, age-dependent downregulation of NMDA receptors (Lethbridge et al. 2012). We did not find significant changes in the physiological actions of NMDA receptors between P9–13 and P18–23, based on measurements of NMDA-evoked slow oscillations (see Fig. 1E), although some specific synapses could show changes in NMDA receptor expression.

Besides providing mechanistic information about age-dependent actions of ARs in OB, our cellular results also help resolve discrepancies between published studies that have examined the acute effect of NE on GC-to-MC transmission. Our prior study (Pandipati et al. 2010), done in P9–13 rats, found that 20 μM NE reduced evoked IPSCs in MCs, while the study of Nai and coworkers (2009), done in P14–30 rats, reported that NE at that concentration increased the frequency of sIPSCs. This difference in part appears to reflect a difference in animal age. We found in the present study that 20 μM NE increased sIPSCs in older animals at P18–23 but had no effect on sIPSCs in P9–13 rats. Differences in the method of measuring GC-to-MC transmission also appear to be important. Here 20 μM NE increased the frequency of sIPSCs in P18–23 rats, as was found by Nai and coworkers in their P14–30 age group, but caused no effect on evoked IPSCs in P18–23 rats. Our study also adds to a growing body of literature examining developmental changes at GC-MC synapses in OB. For example, Dietz and coworkers (2011) measured developmental changes in recurrent dendrodendritic inhibition (DDI) in MCs that likely occurs at GC-to-MC synapses and found, interestingly, that DDI underwent a large reduction over a time frame (between P12 and P20) that roughly matched when we observed a loss of NE-mediated long-term changes in gamma oscillations. Both effects, the reduction in DDI and the loss of the ability to modulate GC-MC synapses, might reflect age-dependent changes in the regulation of GC excitability.

Implications for olfactory function.

At a functional level, what implications might there be for an age-restricted long-term enhancement of gamma oscillations in the OB? Gamma oscillations in the olfactory system appear to be well-correlated with fine odor discrimination (Stopfer et al. 1997). Hence a long-term increase in gamma oscillations in the very young rat could mean that such animals can better discriminate odors compared with prior to the increase. These odors would presumably be ones to which an animal has been previously exposed, in light of the fact that the conditioning stimulus in our experiments that led to long-term enhancement of the gamma oscillations required both NE and neural excitation (Gire and Schoppa 2008; Pandipati et al. 2010). The fact that the plasticity mechanisms appear to be restricted to younger rats at <P14 might reflect the time frame in which rats are first exposed to many key odors to which they need to respond in their lives.

Mechanistically, it is possible that the enhancement of synchronized gamma oscillations improves odor discrimination by increasing the weighting of MC inputs onto downstream pyramidal cells in the olfactory cortex. Enhanced synchrony should increase the summation of excitatory postsynaptic potentials (EPSPs) on these cells. From this perspective, it is notable that the time frame during which the enhanced gamma oscillations occurred in our study, in rats <P14, fits with developmental changes that have been observed at MC-to-pyramidal cell synapses in piriform cortex (Poo and Isaacson 2007). These synapses display typical NMDA receptor-dependent long-term potentiation (LTP) in rats at P5–10 but not at P15–19. Thus two mechanisms may coexist that each support age-restricted enhancement of the pyramidal cell response to MCs. The LTP of MC-to-pyramidal cell synapses would enhance the EPSP in pyramidal cells in response to inputs from any one MC, while an enhancement of synchronized oscillations could increase EPSP summation from multiple MC inputs. It is of course also possible that it is not the long-term enhancement of the gamma oscillations per se that is important for better odor discrimination but other changes for which the gamma oscillations provide a read-out. One example is lateral inhibition in the OB, which, like the gamma oscillations, may depend on interactions between GCs and MCs.

To what extent do our cellular results, showing age-dependent long-term changes in gamma oscillations, fit with reported sensitive periods of olfactory learning? These include associative odor preference learning following classical conditioning, which occurs in rats less than ∼P10–12 (Moriceau et al. 2010; Wilson and Sullivan 1994). Certainly, there are similarities across systems, as our circuit-level changes (in rats <P14) roughly matched in time frame. Also, for both our circuit-level changes (Gire and Schoppa 2008; Pandipati et al. 2010) and associative olfactory learning (Wilson and Sullivan 1994), there appears to be a requirement for a two-component stimulus that includes NE and neural excitation prior to long-term changes. There may, however, be important differences across systems. In our cellular studies α₂-ARs mediated the age-dependent changes in OB circuit properties, while the available evidence indicates that β-ARs are important for odor preference learning (Sullivan et al. 1989, 2000), with a role for α₂-ARs still not well-defined. Moreover, there is evidence that the sensitive period for odor preference learning is caused at least in part by changes in the expression of adrenergic autoreceptors in the locus coeruleus (Moriceau and Sullivan 2004) rather than changes in the OB. It is possible that the cellular effects that we observed are associated with a form of α₂-AR-mediated odor preference learning that remains unidentified, olfactory discrimination learning (Beshel et al. 2007; Doucette et al. 2007; Linster et al. 2002), for which the issue of a sensitive period has not been well studied, or perhaps another form of olfactory learning (Okutani et al. 1999; Shionoya et al. 2006).

GRANTS

This work was funded by National Institute on Deafness and Other Communication Disorders Grants F30 DC-010323 to S. Pandipati, R01 DC-000566 to N. E. Schoppa, and P30 DC004657 to the Rocky Mountain Taste and Smell Center at University of Colorado School of Medicine.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.P. and N.E.S. conception and design of research; S.P. performed experiments; S.P. and N.E.S. analyzed data; S.P. and N.E.S. interpreted results of experiments; S.P. and N.E.S. prepared figures; S.P. drafted manuscript; S.P. and N.E.S. approved final version of manuscript; N.E.S. edited and revised manuscript.