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Published in final edited form as: Neuroscience. 2013 May 30;247:234–241. doi: 10.1016/j.neuroscience.2013.05.040

Olfactory Bulb Monoamine Concentrations Vary with Time of Day

JT Corthell 1,*, AM Stathopoulos 1, CC Watson 1, R Bertram 2, PQ Trombley 1
PMCID: PMC3722297  NIHMSID: NIHMS487259  PMID: 23727009

Abstract

The olfactory bulb (OB) has been recently identified as a circadian oscillator capable of operating independently of the master circadian pacemaker, the suprachiasmatic nuclei of the hypothalamus. OB oscillations manifest as rhythms in clock genes, electrical activity, and odor sensitivity. Dopamine, norepinephrine, and serotonin have been shown to modulate olfactory information processing by the OB and may be part of the mechanism that underlies diurnal changes in olfactory sensitivity. Rhythmic release of these neurotransmitters could generate OB rhythms in electrical activity and olfactory sensitivity. We hypothesized that these monoamines were rhythmically released in the OB. To test our hypotheses, we examined monoamine levels in the OB, over the course of a day, by high-performance liquid chromatography coupled to electrochemical detection. We observed that dopamine and its metabolite, DOPAC, rhythmically fluctuate over the day. In contrast, norepinephrine is arrhythmic. Serotonin and its metabolite HIAA appear to rhythmically fluctuate. Each of these monoamines has been shown to alter OB circuit behavior and influence odor processing. Rhythmic release of serotonin may be a mechanism by which the suprachiasmatic nuclei communicate, indirectly, with the OB.

The olfactory bulb (OB) is one of the few identified circadian oscillators in the brain that is capable of continued rhythmicity without input from the suprachiasmatic nuclei of the hypothalamus (SCN), called the “master circadian pacemaker.” However, the mechanisms that generate the OB’s oscillations are unknown. The purpose of this study was to determine if the content of dopamine (DA), norepinephrine (NE), or serotonin (5-HT) varied across 24 hours and was therefore a possible mechanism underlying OB rhythmicity. Ablation of the SCN affects the period length of OB rhythms (Granados-Fuentes et al., 2004), yet the SCN do not have a known direct connection to the OB. The two structures may interact via OB connection to the lateral hypothalamus (Scott and Pfaffmann, 1967), the SCN connections to the locus coeruleus (LC), the SCN connections to the dorsal hypothalamus that projects to the raphe nuclei (RN), or the SCN connections to the intergeniculate leaflet that projects to the olfactory tubercle and the lateral olfactory tract (for review, see Morin, 2012). Both the OB and the SCN receive input from the RN and the LC. The RN release 5-HT into the OB and SCN, while the LC releases NE into the OB and SCN; the OB cell types and interactions are depicted in Figure 1 (McLean and Shipley, 1987, 1991; Gómez et al., 2005). 5-HT and 5-HT agonists affect SCN rhythms (Prosser et al., 1993) and, in the OB, affect learning and memory (for review, see Fletcher and Chen, 2010) and synaptic transmission (Hardy et al., 2005, Petzold et al., 2009). Rhythmic release of 5-HT into the OB would likely affect how well animals perform on memory tasks as well as affecting OB electrical responses to odorants, depending on the time of day. 5-HT and NE may have additive effects (Yuan et al., 2003), but NE has many varied effects in the OB by itself (for review, see Linster et al., 2011). NE alters odor discrimination and habituation (Guerin et al., 2008), affects learning and memory (Fletcher and Chen, 2010), affects cell death and survival (Veyrac et al., 2005), and affects OB synaptic transmission (Ciombor et al., 1999; Gire and Schoppa, 2008; Jiang et al., 1996; Nai et al., 2010; Trombley and Shepherd, 1992). Rhythmic release of NE by the LC into the OB would alter synaptic transmission and OB circuit dynamics, depending on the time of day that NE was released.

Figure 1.

Figure 1

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Circuit diagram of the olfactory bulb and distribution of monoamines. This diagram shows the layers, primary neuronal populations, and synaptic connections of the olfactory bulb (OB). The white arrows represent excitatory connections; the black arrows represent inhibitory connections. Noradrenergic fibers from the locus coeruleus terminate in the inner plexiform layer (IPL) and granule cell layer (GCL), with lower levels of innervation to the external plexiform layer (EPL) and mitral cell layer (MCL). There is very little noradrenergic input to the glomerular layer (GL). Serotonergic fibers from the raphe nucleus project to all layers of the OB: the densest innervation is in the GL, and the MCL, IPL, and GCL are also densely innervated. The EPL is only sparsely innervated by raphe axons. There are no outside dopaminergic projections to the OB. Intrinsic dopaminergic neurons in the OB consist of a subpopulation of neurons within the GL. OSN (olfactory sensory neuron); PG (periglomerular neuron); T (tufted cell); M (mitral cell); Gr (granule cell).

Both NE and 5-HT could be released into the OB by SCN activity, and both neurotransmitters have been shown to affect SCN and OB synaptic transmission; we, therefore, hypothesized that these two neurotransmitters affect OB rhythms. However, DA also has multiple effects on neurons within the OB and is released by tyrosine hydroxylase-positive juxtaglomerular cells, which are among the targets of 5-HT and NE in the OB (for review, see Cave and Baker, 2009). DA stimulates subventricular zone neurogenesis (Kim et al., 2010), affects odor discrimination (Escanilla et al., 2009), and affects synaptic transmission in the OB (Berkowicz and Trombley, 2000; Davila et al., 2003; Ennis et al., 2001). Rhythmic changes in DA content may alter OB circuit dynamics across the day. Because DA is co-released with gamma-amino butyric acid (GABA; Maher and Westbrook, 2008), outside serotonergic input may activate dopaminergic (DAergic) cells, stimulate DA and GABA release, and decrease glomerular responses to odorants, in addition to 5-HT’s other effects.

We hypothesized that monoamine content in the OB exhibits daily oscillations, distinct from the bulb’s faster electrical oscillations. To test this hypothesis, we used high-performance liquid chromatography with electrochemical detection (HPLC-EC) to measure the monoamine content in the OB. We found that the DA concentration fluctuates over 24 hours, while NE concentration does not. The concentration of 5-HT follows a 10-12 hour rhythm, with nadir 2 hours prior to lights out. These data suggest that OB circuit dynamics, among other behaviors, are altered across the day and cannot be considered static or immune to circadian influences. Additionally, this work supports the idea that the circadian system affects olfactory function (Granados-Fuentes et al., 2011).

1. Experimental Procedures

1.1. Animals

Male and female Sprague Dawley rat pups (Charles River, Raleigh, NC), aged postnatal days 21-23, were kept in standard rat cages under a 12-hr light, 12-hr dark cycle (lights on at 0700 h EST; ZT0). Water and rat chow were available ad libitum. Tissue from 5-7 individual animals was collected at each time point, for a total of 135 animals used for this study. Animals were anesthetized using isoflurane and killed by decapitation. During the night, tissue collection was performed under dim red light. All procedures were approved by The Florida State University Animal Care and Use Committee.

1.2. Tissue Collection

Olfactory bulbs were quickly dissected and placed singly in 1 mL of cold 0.3N perchloric acid to protect against monoamine degradation. Five to seven pairs of olfactory bulbs were collected for every time point. The bulbs were homogenized using a 60 Sonic Dismembrator (Thermo Fisher, Rockford, IL) to release the chemical contents of the tissue sample. Samples were then centrifuged using a 5810 R (Eppendorf, Hamburg, Germany) for 20 min at 6,000 rpm (3,824*g) at 40 C. The supernatant was transferred to a 0.22 μm filter tube and centrifuged again for 10 min at 10,000 rpm (10,621*g). This filtrate was used for analysis by HPLC-EC. The protein pellet was saved for the protein assay. All samples were stored at −80°C until analysis.

1.3. Chemicals

DA, 3,4-dihydroxyphenylaceticacid (DOPAC), 5-HT, 5-hydroxyindoleacetic acid (HIAA), NE, perchloric acid, and 3-methoxy-4-hydroxyphenylglycol (MHPG) were purchased from Sigma Chemical Company (St. Louis, MO).

1.4. HPLC-EC

The mobile phase consisted of 75 mM monobasic sodium phosphate (EMD USA, Rockland, Massachusetts), 1.7 mM 1-octanesulfonic acid (EMD USA), 5-15% HPLC grade acetonitrile (95% purity, EMD USA), and 25 μM of ethylenedinitrilotetraacetic acid, disodium salt (BDH, Radnor, PA). Phosphoric acid was added to balance pH (EMD USA). Water was purified on a Milli-Q system (Millipore, Bedford, MA).

Twenty-five μl of thawed olfactory bulb sample was placed into conical autosampler vials. Twenty μl of each sample was injected by an autosampler (Model 542; ESA, Inc., Chelmsford, MA) and delivered to the column by a Prominence degasser piston pump (LC-20AD; Shimadzu, Kyoto, Japan) and a 100 μl injection loop. The flow rate was set at 0.4 ml/min and the pressure was kept at just below 2000 psi. Monoamine separations were performed using a 150mm x 3.2mm C18 reverse phase column, 3 μm particle size (ESA, Part#70-0636), preceded by a guard column with a pore size of 120 angstroms. The sample was oxidized on a conditioning cell (E, +300mV; ESA 5021) and reduced on an analytical cell (E1, +100mV; E2, −225 mV; ESA 5011). Both channels on the analytical cell were set with a gain of 10 nA, 5 s filter, 1 V output and 0% offset. Changes in current were detected by a Colulochem II detector (ESA) and recorded using EZStart 7.3 SP1 software. All monoamines and metabolites were identified based on peak retention times, and the concentration was measured as the area under the curve versus a standard curve. For detection of MHPG, all samples were heated for 5 min at 94° C to break MHPG-sulfate into free MHPG.

1.5. Protein Assay

The total protein content of each individual bulb was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). Briefly, a standard curve of bovine serum albumin was prepared following kit instructions. Protein pellets were resuspended in 1 ml of PBS. Twenty-five μl of the standard or sample was pipetted, in triplicate, into a 96-well plate. Two hundred μl of working reagent was added to each well. Each plate was left to incubate for 30 min at 37° C. The absorbance of each sample was measured by a V max kinetic microplate spectrophotometer (Molecular Devices, Palo Alto, CA) together with Softmax Version 2.35 software (Molecular Devices) and compared against the bovine serum albumin standard curve. The triplicate measurements were averaged and that average was used to calculate the amount of protein present in the experiment.

1.6. Data Analysis

The data are expressed as a ratio of picomoles (pmol) monoamine per milligram (mg) protein or pmol metabolite/pmol monoamine to reflect turnover and to normalize data. Individual OBs were measured and their contents were averaged between the two bulbs to assess the average per animal. The average amount of monoamine per animal was used for all subsequent data analysis. HPLC-EC data were tested by one-tail ANOVA, using ZT0 as the reference point for the first test (lights on) and ZT12 (lights off) as the reference point for the second test. ANOVA was followed by a Tukey’s Honestly Significant Difference post hoc test, which compared each time point to every other time point. All tests were performed in R (R Core Team, 2012). For all tests, p <0.05 was considered statistically significant and each ANOVA had 23 degrees of freedom.

2. Results

We first examined DA and its metabolite, DOPAC. Because different groups have reported DOPAC/protein (Lookingland et al., 1987) and DOPAC/DA (Hervé et al., 1979) as measures of DAergic activity, we report both here. Figure 2 shows the DOPAC/protein, DA/protein, and DOPAC/DA ratios (A, B, and C, respectively) at hourly time points throughout a day. DOPAC/protein had a single peak at ZT0, when the lights turned on, and DA/protein had multiple peaks throughout the dark and light phases. DOPAC/protein (p = 0.001), DA/protein (p = 0.004), and DOPAC/DA (p = 0.0006) had statistically significant changes as assessed by ANOVA. When we measured NE/protein, two samples stood out. Two of the six samples collected at ZT0 had higher NE/protein ratios than all other samples collected; however, NE/protein did not otherwise fluctuate (Fig. 3). When the two ZT0 outliers were included, NE/protein was statistically significant for ANOVA (p = 0.005). Tukey’s test confirmed that the two animals at ZT0 were the only samples significantly different from the others; when the two outliers were omitted, the ANOVA was not statistically significant, regardless of which reference point was used (p = 0.88). We were unable to acquire consistent readings for MHPG, a metabolite of NE, and so it was not included in our analysis (see Discussion).

Figure 2.

Figure 2

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Dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) time courses. (A) DOPAC/protein as measured by HPLC. Lights are turned on at ZT0 and turned off at ZT12. Dark phase is indicated by gray bar. Points plotted are mean +/− standard error of the mean (SEM). n = 5-7 animals per time point. (B) As A, but DA/protein as measured by HPLC. (C) As A, but DOPAC/DA as measured by HPLC. All measures are reported as picomoles of monoamine per milligram of protein.

Figure 3.

Figure 3

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NE time course. Lights are turned on at ZT0 and turned off at ZT12. Dark phase is indicated by gray bar. Points connected by the line are plotted as mean +/− standard error of the mean. The two points not connected by the line are the two outliers at ZT0. n= 5-7 animals per time point. Measured NE is reported as picomoles of NE per milligram of protein.

HIAA/protein exhibited a large decline between ZT8 and ZT11, producing a twice-daily profile (Fig. 4, A). 5-HT/protein had a similar profile, but with an amplitude of one-tenth the magnitude (Fig. 4, B). Thus, the amplitude of the time course of 5-HT/protein was almost flat relative to that of HIAA/protein, so the HIAA/5-HT profile (Fig. 4, C) reflects the semi-diurnal HIAA/protein profile. HIAA/protein (p < 0.0001), 5-HT/protein (p = 0.0001), and HIAA/5-HT (p < 0.0001) showed statistically significant variation relative to levels at ZT0 and ZT12 by ANOVA.

Figure 4.

Figure 4

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5-HT and hydroxyindole acetic acid (HIAA) time courses. (A) HIAA/protein as measured by HPLC. Lights are turned on at ZT0 and turned off at ZT12. Dark phase is indicated by gray bar. Points plotted are mean +/− standard error of the mean. n = 5-7 animals per time point. (B) As A, but 5-HT/protein as measured by HPLC. (C) As A, but HIAA/5-HT as measured by HPLC. All measures are reported as picomoles of monoamine per milligram of protein.

3. Discussion

We observed that DA and 5-HT fluctuate across the day and that NE does not. This implies that many different OB behaviors are, in the intact animal, affected by the time of day and that DA and 5-HT may be part of the mechanism underlying OB rhythms (Granados-Fuentes et al., 2004; 2011). Both DA and 5-HT fluctuations may change how well animals perform in olfactory tasks, either by DA enhancement of olfactory discrimination (Escanilla et al., 2009) or 5-HT-mediated changes in memory (Fletcher and Chen, 2010) via changes in OB synaptic transmission (Davila et al., 2003; Petzold et al., 2009; Ennis et al., 2001; Berkowicz and Trombley, 2000). Release of DA or 5-HT may also synchronize the OB circadian clock as 5-HT does in the SCN or as DA does in the retina (Ruan et al., 2008). However, other parts of the brain may affect neurotransmitter release into the OB, as melatonin decreases NE release into the pineal gland (Chuluyan et al., 1991) and acetylcholine regulates NE release into the OB (El-Etri et al., 1999). We analyzed DA activity using the DA metabolite DOPAC and not the DA metabolite homovanillic acid (HVA). Some groups have reported that DOPAC is more highly concentrated in some brain regions than HVA (Csernansky et al., 1990; Jenkins, 2008; Natividad et al., 2010), and DOPAC has been previously used as a measure of OB DA activity (Dluzen, 1996; Philpot et al., 1998). Thus, we used DOPAC as our measure of DA activity. However, other groups report higher concentrations of HVA than DOPAC in some brain regions (Gomez et al., 2007; Park et al., 2013; Zant et al., 2011), and one report shows higher HVA than DOPAC in specific brain regions but higher DOPAC than HVA overall (Laatikainen et al., 2013). While it is possible that re-analysis of our data using HVA would alter the amplitude of the observed DA/metabolite ratio fluctuations, our interpretations of the results would be unlikely to change.

The peak in NE appears to be only at ZT0 with a high degree of variance, suggesting that NE is not the synchronizing message from the SCN to the OB. The lack of any NE rhythm also indicates that there may not be a ‘best time’ for NE-mediated olfactory learning. We tried to measure MHPG in our system to have an additional assessment of NE activity in the OB. However, MHPG peaks were not consistent in samples or standards, regardless of mobile phase composition, voltage settings, or run times. Because MHPG standard measurements were not consistent in peak size, elution time, or returning to baseline, we did not pursue MHPG analysis further.

5-HT fluctuations in the OB appear to follow a rhythm of less than 24 hours, and 5-HT release depends on stimulation of the dorsal raphe nuclei; therefore, the dorsal raphe nuclei are likely receiving ultradian input that results in the fluctuations of 5-HT that we report. A report from Gracia-Llanes et al. (2010) indicates that roughly half the synaptic contacts of dorsal raphe axons are with type 1 periglomerular cells (GABA-ergic), as defined by Kosaka and Kosaka (2007). In external tufted cells, 5-HT’s effects are mediated partially by 5-HT2A receptors that activate transient receptor potential channels (Liu et al., 2012), as opposed to the 5-HT2C receptors that mediate SCN and some OB responses to 5-HT (Petzold et al., 2009; Varcoe et al., 2003). Tufted cells also express gap junctions composed of connexin 45 and 5-HT has been shown previously to modulate gap junctional electrical communication via 5-HT2 receptors for connexins 43 and 45 in other systems (Bai et al., 2006; Derangeon et al., 2010; Szabo et al., 2010). Electrical coupling via gap junctions significantly influences OB circuits (Christie et al., 2005; Christie and Westbrook, 2006), however, the effects of 5-HT on gap junctional coupling and communication in the OB are unknown. Taken together, the observed fluctuations in 5-HT may activate periglomerular cells, tufted cells, and cells electrically coupled to tufted cells by gap junctions containing connexin45. Such effects could alter OB responses to odorants at multiple levels of the OB circuit.

DA content appears to fluctuate over time within the OB, with peaks in the light and dark phases. However, DA fluctuations did not synchronize to 5-HT release, indicating that DA rhythms were not the direct result of the reported 5-HT rhythm and presumed 5-HT2A receptor activation. Activation of DAergic neurons and subsequent DA release may be a result of mitral cell rhythmicity, first identified by Granados-Fuentes et al. (2004). If DAergic neurons receive rhythmic inputs, rhythmic activation could synchronize glomeruli that are connected by these cells (Kosaka and Kosaka 2008). DA has been shown to affect the phosphorylation state of gap junctions in the retina, which modulates their ability to provide direct electrical coupling with adjacent neurons (Kothmann et al, 2009). Direct electrical coupling of OB neurons, via gap junctions, contributes to the synchronization of OB neurons projecting to the same glomerulus and is thought to be critical to odor processing by the OB. Whether DA has similar effects in the OB is currently being examined. The difference in light phase/dark phase olfactory sensitivity reported by Granados-Fuentes et al. (2011) may be the result of DA synchronization of the olfactory circuit or 5-HT’s effects on multiple cell types in the olfactory circuit.

4. Conclusions

DA and 5-HT levels in the rat OB fluctuate throughout the day, while NE levels are mostly constant. The 5-HT levels have a semi-diurnal pattern, while the DA fluctuations are less structured. Rhythmic changes in the content and/or release of these neurotransmitters could influence the cellular changes in excitability that underlie circadian rhythms in the OB via modulation of synaptic transmission and electrical coupling.

Highlights.

  • Dopamine content fluctuates in the rat olfactory bulb

  • Serotonin content fluctuates in the rat olfactory bulb

  • Norepinephrine content does not fluctuate in the rat olfactory bulb

Acknowledgements

The authors thank C. Badland for helping us generate the figure artwork, D. Fiore and D. McKee for technical assistance, and J. Olcese for ideas and helpful discussions.

Abbreviations

DA

dopamine

DOPAC

3, 4-dihydroxyphenylacetic acid

HPLC-EC

high-performance liquid chromatography using electrochemical detection

MHPG

3-methoxy-4-hydroxyphenylglycol

NE

norepinephrine

5-HT

serotonin

HIAA

hydroxyindoleacetic acid

SCN

suprachiasmatic nuclei of the hypothalamus

OB

olfactory bulb

LC

locus coeruleus

RN

raphe nuclei

pg

picograms

Footnotes

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Author contributions: JT Corthell and PQ Trombley designed experiments; JT Corthell, AM Stathopoulos, and CC Watson collected samples and performed experiments; JT Corthell analyzed the data and performed statistical analysis; JT Corthell, AM Stathopoulos, R Bertram, and PQ Trombley wrote the manuscript.

Monoamine changes in rat olfactory bulb

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