Title : Non - linear effects of noradrenergic modulation of olfactory bulb function in 1 adult

14 The mammalian main olfactory bulb (MOB) receives a significant noradrenergic 15 input from the locus coeruleus. Norepinephrine (NE) is involved in acquisition of 16 conditioned odor preferences in neonatal animals, in some species-specific odor 17 dependent behaviors and in adult odor perception. We here provide a detailed review of 18 the functional role of norepinephrine in adult rodent main olfactory bulb function. We 19 include cellular, synaptic, network and behavioral data and use computational simulations 20 to tie these different types of data together. 21 22 Introduction 23 24 The locus coeruleus (LC), which releases norepinephrine (NE), is one of several 25 neuromodulatory nuclei with widely distributed ascending projections to cortical brain 26 regions (Foote et al. 1983). LC neurons are activated by novel or target stimuli (Foote et 27 al. 1980; Vankov et al. 1995), and are important in the regulation of vigilance (reviewed 28 in (Aston-Jones et al. 2000; Berridge and Waterhouse 2003). During waking, LC neurons 29 simultaneously fire a brief burst of action potentials in response to non-noxious sensory 30 stimuli of all modalities, but especially those that are novel or salient (e.g., signal reward; 31 (Aston-Jones and Bloom 1981; Aston-Jones et al. 2000). 32 At the neural level, work in somatosensory, auditory, olfactory and visual circuits 33 has revealed complex, often subtle effects of NE on cellular excitability that dramatically 34 alter neuronal responses to sensory input. Some of the better characterized effects 35 include: (1) reduction of spontaneous but not stimulus-evoked firing, thereby increasing 36 signal-to-noise ratios, (2) specific augmentation of stimulus-evoked excitation, and (3) 37 conversion of subthreshold excitatory inputs to suprathreshold spiking responses 38 (Devilbiss and Waterhouse 2004; 2000; Hirata et al. 2006; McLean and Waterhouse 39 1994; Mouradian et al. 1991; Waterhouse et al. 1990). NE also increases the temporal 40 precision between excitatory afferent input and postsynaptic responses, enhancing synchrony 41 between sensory input and spike output (Kossl and Vater 1989; Lecas 2004; Moxon et al. 42 2007). In the main olfactory bulb (MOB) and olfactory (piriform) cortex, NE has a 43 similar potentiating effect on weak sensory inputs as it has in other sensory systems 44 (Bouret and Sara 2002; Ciombor et al. 1999; Hayar et al. 2001; Jiang et al. 1996). NE 45 also decreases response latencies for evoked responses in the MOB and piriform cortex, 46 improving the temporal precision of sensory responses. 47 NE is deeply integrated into the olfactory system. Olfactory cues increase the 48 discharge of LC neurons in behaving animals (Aston-Jones and Bloom 1981) and trigger 49 rapid increases in NE levels in MOB as well as the accessory olfactory bulb (Brennan et 50 al. 1990; Kaba and Keverne 1988; Kaba et al. 1989). LC projections to the AOB are vital 51 for the formation of memories involved in the regulation of pregnancy (Bruce effect) and 52 maternal behavior (Brennan et al. 1990; Kaba and Keverne 1988; Kaba et al. 1989). In 53 neonatal animals, NE-β receptor activation in the MOB plays a critical role in the rapid 54 learning of conditioned odor preferences (reviewed in (Moriceau and Sullivan 2004a). 55 This LC-dependent olfactory imprinting, however, is developmentally transient; while 56 the behavioral effects of bulbar NE modulation in adult rodents have been less studied, 57 they are likely to be more measured and conditional than those in neonates (Moriceau and 58 Sullivan 2004b). More recently increased attention has been paid by several labs to 59 noradrenergic modulation of main olfactory bulb processing in adult animals; we here 60 review these data including cellular, network and behavioral results. We present a 61 comprehensive model of how NE effects on bulbar processing results in the perceptual 62 effects described in the literature. 63 64 Olfactory bulb noradrenergic innervation 65 66 The MOB receives a dense noradrenergic projection from LC that terminates in 67 all but the most superficial layers ((Shipley et al. 1985); Figure 1a). Approximately 40% 68 of LC neurons (400-600 of 1,600 cells) project to the rat MOB (Shipley et al. 1985). NE 69 fibers preferentially target the internal plexiform layer and the granule cell layer, and to a 70 lesser extent the mitral cell layer and external plexiform layer (McLean et al. 1989). 71 Generally paralleling this NE fiber distribution, each of the three major NE receptor 72 subtypes (α1, α2, β) are expressed in multiple layers of the MOB, and individual MOB 73 neurons appear to express multiple NE receptor subtypes (for review, see Ennis et al. 74 2007). For example, mitral cells express all 3 NE receptor subtypes and granule cells 75 express α1 and α2 receptors (Ennis et al. 2007). 76 Physiological considerations suggest that the broad NE fiber innervation of the MOB 77 is paralleled by diffuse NE release in response to LC activation. LC neuronal activity 78 exhibits tonic increases and decreases at the population level in a behavioral state79 dependent manner, and LC neurons fire synchronously in response to excitatory input 80 (Aston-Jones and Bloom 1981; Berridge and Waterhouse 2003). Therefore, even if 81 individual LC axons terminate with sublaminar specificity in the MOB, functionally the 82 parallel activity of the ensemble of LC neurons that project to MOB should lead to 83 diffuse synaptic release of NE. However, there may be laminar differences in 84 concentrations of synaptically-released NE, and perhaps the magnitude of subsequent 85 postsynaptic effects, that are proportional to the density of NE axon terminals. The 86 degree of postsynaptic receptor activation will ultimately depend on the proximity of the 87 receptors to NE terminal release sites. Unfortunately, critical data to assess these issues 88 subcellular localization of noradrenergic receptors in the MOB and their relationship to 89 NE terminal release sites are unavailable. Nevertheless, given these considerations it 90 stands to reason that NE modulation of the MOB network and its impact on olfactory 91 processing cannot be extracted by an understanding of NEs action at a specific receptor 92 or MOB neuronal subtype. Equally importantly, a premise of this review based on 93 emerging and parallel behavioral and electrophysiological findings is that the impact of 94 NE varies dynamically as a function of the extracellular concentration and the 95 constellation of receptors engaged by the prevailing NE level. 96 97 Olfactory bulb network and processing 98 99 The main olfactory bulb in rodents has been extensively described in a number of 100 review articles. We briefly review the main neuronal types, their interactions as well as 101 hypothesized function in odor processing before describing the details of noradrenergic 102 action onto these neurons (Figure 1A). 103 Distributed patterns of activity in response to volatile chemical stimuli (odorants) 104 are transmitted to the olfactory bulb via OSN axons that terminate in the glomeruli of its 105 input layer; each set of OSNs transmits a particular odor receptive field to the glomerulus 106 it projects to (Figure 1A). The olfactory bulb is believed to filter and transform these 107 incoming sensory data, performing normalization, contrast enhancement, signal to noise 108 regulations and other types of operations before conveying the processed olfactory 109 information to several different secondary olfactory structures via mitral/tufted cell axon 110 collaterals (Cleland and Linster 2003). Neuromodulatory inputs such as NE, 111 acetylcholine and serotonin modulate and change these functions of the olfactory bulb, 112 presumably adapting the performed computations to maximize processing dependent on 113 the specific behavioral demands on the animal (reviewed in (Mandairon and Linster 114 2009)). 115 Concentration invariance or normalization of odor representations is a function 116 which has been proposed for bulbar processing (Cleland et al. 2007); this function may 117 rely on the dense glomerular networks of external tufted, short axon and periglomerular 118 cells described by Aungst et al. (Aungst et al. 2003) (Figure 1B&C). Cholinergic and 119 serotonergic inputs acting on sub-populations of these neurons could modulate the 120 processing leading to concentration invariant odor representations. 121 Contrast enhancement is a common property of sensory systems that narrows 122 (sharpens) sensory representations by specifically inhibiting neurons on the periphery of 123 the representation, thus enhancing the contrast between signal and background (Figure 124 1B). Contrast enhancement of odor representations in the olfactory bulb is thought to be 125 mediated by inhibitory neurons in both the glomerular layer (periglomerular cells, 126 (Cleland and Sethupathy 2006; Linster and Gervais 1996; Linster and Hasselmo 1997)) 127 and granule cell layer (granule cells, (Urban 2002; Urban and Arevian 2009; Yokoi et al. 128 1995)). A number of computational models have proposed solutions for this important 129 function, including lateral interactions between glomeruli (Linster and Gervais 1996; 130 Linster and Hasselmo, 1997), computations local to each glomerulus (Cleland and 131 Sethupathy 2006) (Figure 1B&C) and local and lateral interactions between mitral and 132 granule cells (Urban and Arevian 2009) (Figure 1B&C). Modulatory inputs acting on the 133 excitability and synaptic interactions in these bulbar layers could therefore change these 134 computations and modulate the degree of contrast enhancement performed in a given 135 behavioral situation. Behavioral data in which modulatory functions in the MOB have 136 been disturbed clearly point to contrast enhancement being an important function of the 137 MOB (reviewed in (Mandairon and Linster 2009). For example, enhancing cholinergic 138 activity in the MOB increased rats’ ability to discriminate between chemically very 139 similar odorants, whereas blockade of cholinergic receptors decreased this 140 discrimination; these results can be predicted by known effects of cholinergic inputs onto 141 glomerular circuits mediating contrast (Chaudhury et al. 2009; Linster and Cleland 2002; 142 Mandairon et al. 2006a). 143 At the level of glomerular processing, changes in activation patterns, i.e., which 144 mitral cells are responsive to any given odorant are thought to be dominant and the 145 functions described above modulate these activation patterns. In deeper layers, 146 modulation of spike timing and synchronization may affect contrast of odor 147 representations (Figure 1B&C), as the dynamics of bulbar processing seem to be mainly 148 regulated by the interactions between mitral and granule cells. Modulatory inputs on 149 mitral and granule cells can potentially affect the dynamics of neural activity as well as 150 spike timing and degrees of synchronization. Levels of synchronization among bulbar 151 outputs have been proposed to contribute to odor processing and learning experimentally 152 (Beshel et al. 2007; Brea et al. 2009; Kay et al. 2009; Nusser et al. 2001). Modulatory 153 inputs to granule and mitral cells can potentially regulate oscillatory dynamics of 154 olfactory bulb processing and hence change the processing of odors, as proposed early on 155 by Freeman and colleagues (Di Prisco and Freeman 1985; Gray et al. 1986) and shown 156 recently in slices of young rats by Schoppa and colleagues (Gire and Schoppa 2008; 157 Pandipati et al. 2010). 158 In summary, modulatory inputs have the capacity to regulate bulbar processing at 159 many levels and have been shown to do so in a variety of paradigms and levels of 160 investigation: glomerular modulation regulates contrast and normalization processing 161 whereas deeper layer modulation regulates contrast, dynamics and synchronization 162 properties. In the next section, we will review data pertaining to the noradrenergic 163 modulation of these different functionalities and summarize these data with a 164 comprehensive model of bulbar processing and noradrenergic modulation of this 165 processing. 166 167 168 169 Noradrenergic modulation of bulbar processing: electrophysiological 170 evidence 171 Because this review is meant to focus on noradrenergic modulation of adult 172 rodent olfaction, we will focus the review of cellular and synaptic effects on those data 173 obtained in young to adult rodents. Data from neonatal rodents and data from other 174 species will be discussed when necessary to make a specific point only but will not be 175 extensively reviewed here. As extensively reviewed by Sullivan and colleagues, 176 noradrenergic modulation and the expression of noradrenergic receptors in the olfactory 177 bulb of rodents changes dramatically after postnatal days 9-10 in rats (Moriceau and 178 Sullivan 2004b; Sullivan and Wilson 2003). The role of noradrenergic modulation in 179 odor learning as well as the receptors involved in learning change after the critical period; 180 this behavioral observations has also been confirmed in brain slice recordings: there is a 181 marked increase in the density of β receptor binding in the rat olfactory bulb from 182 postnatal day 12 to 30 (Woo and Leon, 1995); the expression of mRNA for the α2A 183 receptor subtype increases until postnatal day 21, but levels are lower in the adult 184 olfactory bulb (Winzer-Serhan et al. 1997); and the effects of NE as well as the specific 185 receptors involved in these effects differed in slices obtained in younger (P9-13) or older 186 (P19-21) rats (Pandipati et al. 2010). For sake of consistency and focus on adult 187 perception and learning, we will therefore only discuss results obtained in animals after 188 the critical learning period, while only briefly mention results in younger animals. 189 190 Modulation of mitral cell responsiveness to low amplitude sensory stimuli. 191 Activation of LC in anesthetized rats enhanced mitral cell spiking responses to 192 weak (i.e. perithreshold) but not strong (i.e. suprathreshold) stimulation of the olfactory 193 epithelium (Jiang et al. 1996). The LC-evoked enhancement occurred without consistent 194 changes in mitral cell spontaneous firing rate, although a subset of cells showed a small 195 decrease in activity. Consistent with these in vivo findings, application of low 196 concentrations of NE (1 μM) selectively increased mitral cell spike discharge to 197 perithreshold intensity olfactory nerve (ON) stimulation in rat MOB slices (Ciombor et 198 al. 1999). NE also decreased the inhibitory period following ON stimulation-evoked 199 spikes. The NE response enhancement was due to a reduction of spike response failures 200 to ON stimulation rather than an increase of the incidence of evoked-spikes for threshold 201 ON stimuli. Thus, NE converts a previously subthreshold excitatory synaptic response 202 into a suprathreshold, spike-triggering response, similar to the effect of NE at α1 203 receptors in the cortex (Mouradian et al. 1991; Waterhouse et al. 1990). The perithreshold 204 ON response enhancement, but not the decrease in inhibition, was mimicked by α1 205 receptor agonists although the magnitude of the enhancement was less than that for NE 206 (Ciombor et al. 1999; Hayar et al. 2001). This indicates that the effect of NE may involve 207 multiple noradrenergic receptors. Taken together, these findings suggest that NE release 208 alters mitral cell excitability in a manner that could increase their sensitivity to weak or 209 subthreshold ON input, perhaps to improve the detection of weak odorants. The onset 210 latency for ON-evoked spikes was significantly reduced by NE, thereby more closely 211 synchronizing mitral cell output to sensory input, similar to NE actions in a number of 212 cortical circuits including olfactory cortex (Bouret and Sara 2002; Devilbiss and 213 Waterhouse 2000; Waterhouse et al. 2000). 214 215 Modulation of mitral cell responsiveness to odors. 216 The impact of NE on cellular responses to odors has been examined in only few 217 studies. Repetitive pairing of LC stimulation and odors producing excitation of a mitral 218 cell was found to produce a long-lasting (4 hr) suppression of subsequent responses of the 219 cell to the paired odor in anesthetized mice (Shea et al. 2008). This suppression was 220 prevented by combined blockade of α and β receptors near the recorded mitral cell. 221 Behavioral testing 24 hr after such pairing indicated that mice investigated the paired 222 odor less than an unpaired odor, suggesting that LC stimulation-odor pairing produces a 223 memory expressed by decreased interest specific for the paired odor. LC-induced 224 suppression of mitral cell excitatory odor responses, when put into context with the 225 preceding studies showing that LC stimulation or NE application enhances mitral cell 226 responses to perithreshold intensity ON stimulation, show that NE modulation is not a 227 simple effect independent of time course or experimental protocol. Differences in 228 species, the duration or magnitude of LC activation as well as the mitral cell sensory 229 response may be important factors; however, behavioral studies in mice suggest that NE 230 may play an important role in behavioral habituation to odorants (Guerin et al. 2008), 231 these data will be discussed in more detail below. 232 233 234 Modulation of mitral and granule cell properties. 235 Historically, findings obtained in turtle and cell cultures of neonatal rats led to the 236 conclusion that a major action of NE is mitral cell disinhibition subsequent to inhibition 237 of, or decreased GABA release from, granule cells (Jahr and Nicoll 1982 (Trombley 238 1992; Trombley and Shepherd 1992). Subsequent studies in juvenile and adult rat MOB 239 slices demonstrated noradrenergic agonists did not effect ON stimulation evoked field 240 potentials (FPs) recorded in the glomerular layer, or ON stimulation evoked postsynaptic 241 currents in mitral cells (Hayar et al. 2001). This suggests that NE-evoked enhancement of 242 ON stimulation evoked mitral cell spiking is mediated by actions on MOB neurons. 243 Interestingly, a relatively high concentration of NE (30 μM) suppressed ON stimulation244 evoked FPs and EPSCs by activating inhibitory presynaptic D2 dopamine receptors on 245 ON terminals. Thus, high concentrations of NE may have non-specific inhibitory effects 246 that dampen ON input. Since low concentrations of NE or α1 receptor agonist did not 247 modulate the strength of ON input, how does NE and α1 receptor activation enhance 248 mitral cell responses to weak ON input observed in extracellular studies (Ciombor et al. 249 1999; Jiang et al. 1996)? One possibility is that NE (1-30 μM), acting at α1 receptors, 250 directly depolarizes and induces an inward current in mitral cells (Hayar et al. 2001). This 251 effect was mediated by closure of a leak potassium current as determined by the reversal 252 potential and increased membrane resistance associated with the inward current. These 253 results indicate that NE-induced activation of the α1 receptor produces a small 254 depolarization that moves the mitral cell membrane potential towards spike threshold, 255 while the resistance increase boosts somatic depolarization to synaptic inputs. Together, 256 these effects cooperate to increase the probability of spike initiation in response to weak 257 ON synaptic input (Hayar et al. 2001). 258 Individual MOB neurons appear to express multiple NE receptor subtypes which 259 have differing affinities for NE (Ramos and Arnsten 2007). Therefore, the effect of NE 260 on MOB neurons and the MOB network is likely to vary as a function of the extracellular 261 concentration of NE. To investigate this question, the effects of varying NE 262 concentrations on GABAergic inhibition of mitral cells as assessed by spontaneous IPSC 263 activity was examined in rat MOB slices (Nai et al. 2009). The lowest concentrations of 264 NE used reduced IPSC frequency with further increases in concentration producing an 265 inverted-U profile characterized by an intermediate dose increase in IPSCs and a high 266 dose decline in IPSCs towards baseline level. Specifically, NE concentrations <1 μM 267 suppressed IPSCs, whereas 1-30 μM increased IPSCs; concentrations above 30 μM 268 reversed the facilitation. These effects were presynaptically mediated by NE actions on 269 GABAergic inputs to mitral cells. As determined by subtype specific agonists and 270 antagonists, the low concentration suppression was mediated by the α2 receptor, while 271 the intermediate concentration facilitation was mediated by α1 receptors (Nai et al. 272 2009). At the intermediate NE levels, the α1 receptor stimulatory effect appears to 273 overrides the α2 receptor inhibitory effect on GABA release (Figure 2A&B). 274 The concentration profile for the effects of NE on the level of GABAergic 275 inhibition of mitral cells was well matched by direct effects of NE on granule cell 276 excitability in rat MOB slices. Specifically, low concentrations of NE (0.1-1.0 μM) or α2 277 receptor agonists hyperpolarized and inhibited granule cell spontaneous or evoked spike 278 discharge (Nai et al. 2010). By contrast, 10 μM NE or α1 receptor agonists depolarized 279 and increased granule cell discharge. These effects appear to be mediated by opposing 280 actions on granule cell potassium currents, with α2 receptor activation increasing, and α1 281 receptor activation inhibiting, potassium currents (Nai et al. 2010). These studies indicate 282 that the differential affinities of NE receptor subtypes allow for differential modulation of 283 GABA release and olfactory processing as a function of the level of NE release, which in 284 turn, is regulated by behavioral state (Berridge and Waterhouse 2003). 285 Relevant to the preceding studies is the consideration of how the concentration 286 range of exogenously applied NE correlates with levels of synaptically released NE. In 287 anesthetized rats where LC spontaneous discharge rate averages 1-2 Hz, basal 288 extracellular concentrations of NE in the rat olfactory bulb measured by in vivo 289 microdialysis are 0.3-0.4 nM (El-Etri et al. 1999); tonic increases in LC discharge to 14 290 Hz elicited by intra-LC microinjection of acetylcholine elevated NE levels to ~1.0 nM. 291 However, NE levels at synaptic release sites may be substantial higher than values 292 obtained via in vivo microdialysis. Also, microdialysis samples are collected over many 293 minutes and may not accurately reflect phasic or peak changes in NE levels. More 294 sensitive electrochemical measurements of phasic NE release in the cerebellum of 295 anesthetized rats indicate that 10 Hz LC electrical stimulation increases NE levels to 1-2 296 μM (Bickford-Wimer et al. 1991). The later value is compatible with the effects of sub297 μM to low μM concentrations of bath-applied NE on mitral cell IPSCs and ON 298 stimulation-evoked spiking summarized above. Unfortunately, detailed measurements of 299 basal and phasic, LC-evoked NE levels in the olfactory bulb in unanesthetized rats are 300 unavailable. 301 302 Resulting modulation of network properties. 303 The combined effects of NE receptor activation on bulbar cell types results in complex 304 and concentration dependent effects on network processing reviewed below. NE infusion 305 (100 μM) into the MOB of awake rabbits increased the amplitude of gamma (40-80 Hz) 306 components of the MOB EEG and also potentiated the spatial EEG pattern elicited by 307 novel odors (Gray et al. 1986). In the same preparation, β receptor antagonist infusion 308 prevented transient alterations of the MOB EEG elicited by repetitive presentation of a 309 reinforced, but not unreinforced, odor (Gray et al. 1986). These observations can be 310 ascribed to the increased excitation of both mitral and granule cells (see previous section 311 for details) leading to a stronger feedback coupling between these cell types which 312 naturally results in higher oscillatory power (Figure 3A). 313 Several studies have investigated the effects of NE on the well characterized field 314 potential evoked by stimulation of the lateral olfactory tract (LOT) which antidromically 315 activates mitral cells and engages the mitral cell to granule cell synapse. LC stimulation 316 was reported to have no effect on LOT-evoked FPs recorded in the rat granule cell layer 317 (Perez et al. 1987). A subsequent study in rat using a paired-pulse LOT stimulation 318 protocol reported that LC activation had no effect on the conditioning pulse-evoked FP in 319 the granule cell layer. However, LC stimulation elicited an initial phasic decrease 320 followed by a long-lasting increase in paired-pulse depression of the EFP (Okutani et al. 321 1998). This biphasic effect, attributed to β receptor activation, was interpreted as an NE 322 elicited reduction, then facilitation of granule cell-mediated inhibition of mitral cells. 323 Another study reported that NE infusion (0.1-1.0 mM) into the rat MOB, acting at α1 but 324 not α2 or β receptors, increased the LOT conditioning pulse-evoked granule cell layer 325 FP; mitral cell antidromic responses were unaffected, suggesting that NE increases 326 synaptic activation of granule cells (Mouly et al. 1995), a finding compatible with 327 increased excitation of granule cells. In summary, the studies above indicate that LC 328 stimulation or NE infusion: (1) has no effect on mitral cells or granule cells (Perez et al. 329 1987); (2) biphasically inhibits then facilitates granule cell-mediated inhibition via β 330 receptors (Okutani et al. 1998); and (3) facilitates activation of granule cells and 331 subsequent granule cell-mediated inhibition via α1 receptors (Mouly et al. 1995). It is 332 important to note that interpretation of paired-pulse tests at mitral cell-granule cell 333 reciprocal dendrodendritic synapses are especially problematic if NE simultaneously acts 334 on preand postsynaptic sites under study. 335 336 Noradrenergic modulation of olfactory bulb processing 337 As described in the previous sections, NA has multiple effects on bulbar cellular 338 and synaptic parameters in the olfactory bulb and these effects are strongly concentration 339 dependent. Taken together these individual actions lead to changes in network processing 340 which can be difficult to predict due to the non-linearity of cellular processes and the 341 differential binding affinities of the receptors involved (Table 1). Interestingly, despite 342 the fact that all NE receptor types have been localized across the glomerular layer, no 343 data as to modulation of glomerular processing by NE inputs is available. Hence, the 344 following functional discussions will focus on deeper layer processing. One must keep in 345 mind that inconsistencies between electrophysiological findings and behavioral data can 346 potentially be explained by the missing information pertaining to glomerular modulation 347 by NE. As detailed above it is believed that contrast enhancement functions for example 348 are performed in the glomerular layer, hence any effect NE would have on the networks 349 involved would modulate odor perception and learning. 350 Figure 2 illustrates the effects of NE activation of α1 and α2 receptors on 351 simulated granule and mitral cells. Because NE activation of α1 and α2 receptors creates 352 opposite effects on granule cell activation, low concentrations of NE decrease granule 353 cell firing (Figure 2A; simulated by increased spiking threshold) and with it mitral cell 354 inhibition whereas at higher concentrations, α1 effects are stronger and granule cell 355 activation is increased (Figure 2A; simulated by decreased firing threshold) resulting in 356 stronger inhibition of mitral cells. The net effect, shown as changes in granule cell 357 spontaneous firing in Figure 2B, with magnitudes comparable to those recorded in slices 358 (Nai et al. 2009) is that of a decrease followed by an increase in firing. In addition to the 359 modulation of granule cell firing, activation of α1 receptors depolarizes mitral cells 360 (simulated by a change in membrane depolarization; Figure 2C) and hence 361 counterbalances the effects of increased inhibition on this particular response. As a 362 consequence, mitral cell responsiveness to very low concentration odor stimuli increases 363 with increasing NE concentrations despite increasing tonic inhibition on mitral cells 364 (Figure 2D). At higher NE concentrations responses to weak stimuli are decreased with 365 respect to the peak response due to the additive effects of α1 and α2 receptor activation 366 (Figure 2D). This particular combination of effects could explain why low odor detection 367 is improved by bulbar NE without affecting odor discrimination capabilities to the extent 368 predicted by increased responsiveness of mitral cells alone (Figure 3). Figure 3A-C show 369 examples of simulated network responses during spontaneous and odor-elicited activity 370 in a simulation of olfactory bulb. These figures illustrate the modulation of network 371 properties based on changes in cellular properties in a computational model of olfactory 372 bulb processing. Changes in signal-to noise ratio are highlighted by comparing the 373 magnitude of stimulus-responses to spontaneous activities and changes in dynamics are 374 highlighted by comparing stimulus-evoked field potential oscillations in the three cases. 375 The network was stimulated with very low odor concentrations (best OSN response was 376 10% of maximally possible response) and no NE, low NE (α2 only) and high NE (α1 + 377 α2) modulation was simulated using the cellular parameters shown in Figure 2. At low 378 NE concentrations granule cell spontaneous activity decreases and mitral cell 379 spontaneous activity is slightly higher than under control conditions. However mitral cell 380 responsiveness to odor stimulation is increased; as a consequence the overall signal-to381 noise ratio increases. At higher NE concentrations, increased granule activity decreases 382 mitral cell spontaneous activity, during odor stimulation this effect is counteracted by the 383 a1 mediated increase of mitral cell responsiveness. In combination these two effects lead 384 to a net increase in signal-to-noise ratio as compared to control. Overall, the relative 385 response to odor compared to spontaneous activity is enhanced, as described previously 386 in detail (Hasselmo et al. 1996, Escanilla et al. 2010). 387 Simulation of NE modulation in a computational model of olfactory bulb 388 processing showed that: (1) activation of granule and mitral cells by α1 receptor 389 activation increases oscillations and synchrony among olfactory bulb mitral cells, (2) the 390 combination of granule and mitral cell activation leads to enhanced responses to low 391 threshold stimuli and (3) signal-to-noise ratio in the model was improved in mitral cells 392 in response to odor stimulation (Escanilla et al. 2010). In the model these effects were 393 prominent when NE concentrations were modeled to be in the range of α1 receptor 394 activation (Figure 3C). In the low concentration range, α2 receptor activation dominates 395 and leads mainly to a small increase in mitral cell activation due to a decrease in granule 396 cell inhibition (Figure 2C&D). Overall, odor detection, calculated as the distance between 397 spontaneous and odor activated activity increases and then decreases as NE levels in the 398 model increase (Figure 4A). The non-monotonic curves qualitatively follow those of the 399 combination of α1 and α2 effects at the cellular level (Figure 2B&D). Discrimination 400 between two highly overlapping odorants, calculated as the distance between odor 401 evoked network activities increases with increasing NE concentration. Figure 3 illustrates 402 that while stimulus evoked responses are more easily detected at low NE concentration, 403 the responses to two different, but overlapping odorants are more apparent at higher NE 404 concentration. The increase in discrimination is due to increased inhibition paired with 405 depolarization in mitral cells in response to α1 activation mainly (Figure 4B). In addition 406 to modulation of rate-based activation patterns used to calculate these distances, 407 oscillatory power and synchronization between mitral cells is also modulated by levels of 408 NE inputs in the model as shown previously in (Escanilla et al. 2010) (Figure 3, 4D). 409 The computational model also puts into evidence that activation of α1 receptors in mitral 410 cells alone, while enhancing odor detection, would not increase discrimination (Figure 411 4C). If noradrenergic depolarization of mitral cells is accompanied by increased 412 inhibition from granule cells through NE, the net result is enhanced detection by 413 lowering mitral cell response threshold, as well as enhanced discrimination – by 414 increasing inhibition and making weakly activated mitral cells silent. While NE 415 depolarization of mitral cells alone entrains granule cells and leads to increased inhibition 416 of mitral cells, in the model used here this is not sufficient to offset the NE depolarization 417 of mitral cells in order to obtain good odor discrimination. When NE activates only one 418 cell type the system becomes unbalanced and the result is an enhancement of detection 419 accompanied by low discrimination. The simultaneous activation of mitral and granule 420 cells also leads to enhanced oscillations which increase signal-to noise ratio as well as 421 spike synchronization among mitral cells (Figure 3). 422 423 Behavioral results 424 Olfactory perception and learning can be measured with a number of different 425 paradigms in rodents, such as habituation, perceptual learning, discrimination learning 426 and social interactions. Because of the choice of paradigms, odors, concentration and 427 other variables, it is relatively difficult to directly compare results from different studies. 428 It is therefore useful to consider these studies in the light of the previously discussed 429 cellular, synaptic and network effects of noradrenergic modulation in order to interpret 430 them within a common framework. 431 A study by Royet et al (Royet et al. 1983) showed that food deprived rats with 432 lesions of noradrenergic inputs to the olfactory system were more likely to eat novel 433 foods than control rats. The authors concluded that NE modulates the bulbar response to 434 food odors. A follow up study showed that in rats with similar lesions, mitral cell 435 responses to food odorants were enhanced (Gervais and Pager 1983). In the light of 436 previously discussed cellular effects, mitral cell excitability could be increased because of 437 missing noradrenergic excitation of granule cells, leading to higher excitability in mitral 438 cells. 439 With respect to general odor detection, a recent study by Escanilla et al. (Escanilla 440 et al.) showed that rats infused with additional NE into their olfactory bulb detected 441 odorants at substantially lower concentrations than saline infused control rats. Given that 442 NE increases mitral cell excitability in vitro as well as in vivo and increases responses to 443 weak olfactory nerve stimuli, these behavioral data support the idea that bulbar NE 444 increases the sensitivity of the MOB to low intensity stimuli. The behavioral NE effect 445 was mainly mediated by α1 receptors in these studies: blockade of α1 receptors 446 abolished the effect of NE infusions; these data are in agreement with the brain slice 447 results discussed above (Nai et al. 2009; Nai et al. 2010). 448 Habituation, a commonly used paradigm to test olfactory perception and memory 449 in rodents, has been reported to be affected by manipulations of the noradrenergic 450 system. In a habituation paradigm, animals form a memory to an odorant to which they 451 are repeatedly exposed. The duration and specificity of this memory can be assessed by 452 presenting the same or novel odorants after memory formation. Guan et al. (Guan et al. 453 1993b) reported that in mice in which noradrenergic inputs to the MOB were lesioned via 454 6-ODHA infusions, the discrimination of general odorants after habituation – i.e., the 455 specificity of the habituation memory was impaired. In contrast, discrimination between 456 social cues was not affected: general lesions of LC neurons decreased investigatory 457 responses to urinary odorants in mice but did not change the discrimination of different 458 urinary cues (Guan et al. 1993a; Guan et al. 1993b). This result was contradictory to a 459 later study showing a general inability to form the habituation memory after DSP-4 460 lesions in mice, albeit in this study general odorants instead of urinary stimuli were used 461 and NE depletion was not specific to the olfactory bulb (Guerin et al. 2008). In the latter 462 study habituation to an olfactory stimulus was restored in DSP-4 lesioned mice when NE 463 was reintroduced bilaterally into the MOBs, strongly suggesting that bulbar NE is 464 important for the formation of a habituation memory (Guerin et al. 2008). These data are 465 supported by later electrophysiological data showing strong adaptation of mitral odor 466 responses when odor stimulation was paired with stimulation of LC neurons (Shea et al. 467 2008). 468 In agreement with Guan et al, the role of bulbar NE in discrimination of odorants 469 has also been shown in experiments by Mandairon et al (1996) in which bulbar NE 470 receptors were specifically blocked; in these experiments the specificity of the formed 471 habituation memory was decreased when α1, but not α2 or β receptors were blocked in 472 the MOB (Mandairon et al. 2008). Escanilla et al (2010) support these data by showing 473 an increase in odor discrimination at relatively low odor concentrations when NE is 474 added to the MOB (Escanilla et al. 2010). In summary, all experiments using a 475 habituation-dishabituation paradigm to test odor discrimination show that bulbar NE 476 modulates discrimination after memory formation. Modulation of discrimination between 477 perceptually similar odorants can be predicted from the combination of increased 478 excitability and increased inhibition evidenced in brain slice experiments and put into 479 context by computational modeling of these results. The results pertaining to the 480 formation of the habituation memory are contradictory and may be due to differences in 481 the behavioral paradigms used, specifically the time course of odor presentations which 482 has been shown to be modulated by NE (Veyrac et al. 2007). 483 In contrast to the spontaneous discrimination between odorants tested when a 484 habituation task is used, discrimination in a rewarded force choice task was only affected 485 when all NE receptors were blocked: similar results were obtained in two very different 486 behavioral paradigms in mice (Doucette et al. 2007) and rats (Mandairon et al. 2008). 487 The former study showed that in an automated successive go-no-go task, mice with all 488 bulbar NE receptors blocked were significantly impaired in learning a discrimination 489 between highly similar binary mixtures only. Discrimination of “easy” odor pairs was not 490 affected in this experiment (Doucette et al. 2007). Similarly, in a naturalistic 491 simultaneous forced choice discrimination task, rats’ ability to discriminate perceptually 492 very similar odorants was impaired when all bulbar NE receptors were blocked, but not 493 when individual receptors were blocked (Mandairon et al. 2008). 494 In summary, current odor discrimination experiments in adult rodents support the 495 idea that NE changes odor processing in the MOB in such a manner as to support specific 496 odor representations and to facilitate the separation of representations with highly similar 497 input patterns. At low odor concentrations and in a spontaneous discrimination task, α1 498 receptors seem to be mainly involved in this process whereas in a reward motivated task 499 all three receptor types interact. Figure 5 summarizes behavioral results that are in 500 agreement with the computational results shown in Figure 4. Odor detection and 501 discrimination at very low stimulus concentrations (10-4 Pa) are graphed as a function of 502 NE concentration infused into the OB during the behavioral task. Note that the magnitude 503 of detection and discrimination is not a mono-tonic function of NE concentration, as 504 would be predicted from the non-monotonic effects on cellular parameters discussed 505 above. An increase in discrimination of odorants with similar bulbar representations 506 could be achieved by (1) increased granule cell excitability leading to inhibition of 507 weakly activated mitral cells (2) increased mitral and granule cell excitability leading to 508 stronger feedback and as a consequence higher oscillatory power and synchronization 509 between responsive mitral cells. This effect would be mainly through α1 receptors as 510 described above. In a reward driven discrimination task the animals are highly motivated 511 and one can speculate that higher order structures are involved in processing of odor512 reward associations. Blockade of all three receptor types is necessary in order to impair 513 rats or mice in a reward-motivated task, suggesting that bulbar processing differs between 514 spontaneous and reward-motivated tasks or that the downstream processing of bulbar 515 information is different (Doucette et al. 2007; Mandairon et al. 2008). This apparent 516 difference in the bulbar role across tasks has been observed in a number of experiments. 517 For example, habituation memory was not affected by broad lesions of feedback to the 518 MOB from central structures whereas learning of a reward-motivated discrimination was 519 (Kiselycznyk et al. 2006). Blockade of cholinergic receptors in the MOB, individual or 520 global, in contrast, only affected habituation memory but not discrimination learning in a 521 reward-motivated task (Chaudhury et al. 2009; Linster et al. 2001; Mandairon et al. 522 2006a). The effects of different modulators may be a means to distinguish between the 523 role the MOB plays in different types of behaviors relying on olfaction. 524 525 Discussion 526 The olfactory system is an ideal model to study sensory modulation by NE inputs. 527 First, the MOB has a dense NE innervation, second it is accessible to experimentation at 528 many levels, third it is relatively segregated from other brain structures and fourth, it’s 529 inputs and outputs are well characterized. Brain slice experiments in combination with 530 behavioral pharmacology and computational modeling show that it is the combination of 531 NE effects on mitral and granule cells that creates the behaviorally observed modulation 532 of odor detection and discrimination (Figure 6). Activation of α1 and α2 receptors create 533 non-linearities due to the relative affinities of these receptors. How these non-linearities 534 are behaviorally relevant needs to be further investigated. Effects of β receptors have not 535 yet been determined in behavioral studies other than the fact that effects on rewarded 536 odor discrimination tasks can only be observed when all receptor types are blocked. 537 Further brain slice experiments elucidating NEs effects at β receptors are needed to 538 design specific behavioral experiments to test the role of this receptor in adult rodent odor 539 processing. Thus far, results on the role of NE in early olfactory processing are in good 540 agreement with results from other systems: a modulation of signal-to-noise ratio, 541 detection of low amplitude stimuli and discrimination between similar stimuli. 542 In general, converging data from electrophysiological studies of cellular543 membrane properties of MOB neurons and behavioral assays of perception reviewed here 544 indicate that NE biases the MOB network to improve odor detection and discrimination. 545 Despite these advances, many questions about NE function in the MOB remain. The 546 cellular effects of NE on other MOB neuronal subtypes such as tufted cells and non547 granule cell interneurons, as well as if and how NE modulates centrifugal input from 548 olfactory cortical structures, are unknown. Such studies will be critical to better 549 understand how NE modifies the MOB network. In other systems, NE is known to alter 550 network properties in a manner to sharpen stimulus representation. In visual cortex, NE 551 enhances selectivity for stimulus speed and direction (McLean and Waterhouse, 1994). In 552 the cochlear nucleus and auditory cortex, NE refines receptive fields, increasing 553 selectively for pure-tones (Manunta and Edeline, 1999; Manunta and Edeline, 2004). In 554 the thalamus, NE refines whisker receptive fields to the most strongly tuned or principal 555 whisker (Hirata et al., 2006). While cellular and behavioral data suggest that NE may 556 impact similarly on mitral cell odor receptive fields, recordings of odor responses during 557 LC activation or NE infusion are necessary to evaluate this possibility. 558 559 Acknowledgements: Supported by PHS Grant CRCNS DC008702 to CL and ME. 560 561 562 563 564 Aston-Jones G, and Bloom FE. 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Within this glomerulus, OSNs make excitatory synapses onto mitral and tufted 763 cells, the primary output neurons of the MOB, as well as glomerular layer interneurons 764 comprising periglomerular (PG) and external tufted (ET) cells. Most PG cells (~70%), 765 and a third type of interneuron, short axon cells (SA), are not directly activated by OSNs. 766 PG, ET and SA cells form intricate networks within the glomerular layer which have 767 been proposed to perform operations such as normalization, contrast enhancement and 768 synchronization. In deeper processing layers mitral cells (Mi) interact with at least one 769 other class of interneurons, granule cells (Gr). These provide extensive feedback and 770 lateral interactions between mitral cells by interacting with their elongated secondary 771 dendrites. This layer of processing is thought to be involved in creating olfactory bulb 772 gamma rhythms and generating synchronized spike patterns. Noradrenergic inputs from 773 the locus coeruleus activate three classes of noradrenergic receptors distributed across the 774 MOB. α1 receptors are thought to be predominantly located on Mi and Gr cell bodies as 775 well as secondary dendrites with a sparser distribution in the glomerular layers; α2 776 receptors are mainly located on granule cells with a sparse distribution on Mi cell bodies 777 and the glomerular layer; β receptors have been reported on Mi cell bodies and in the 778 glomerular layer. B. Schematic depiction of glomerular input layer and Mi cell activity 779 patterns in response to odor stimulation. Bi-Biii show simulated distributed odor responses 780 at two concentrations with lower concentration in the left column and high concentration 781 in the right column. The 2-dimensional simulations are color coded with red indicating 782 high and dark blue low levels of activity. Bii shows the same patterns after amplitude 783 invariance processing has been performed by the network of local interneurons and Biii 784 shows the same pattern after contrast enhancement. The patterns in Biii represent the end 785 result of the glomerular computations transmitted to deeper layers by Mi cells. The 786 details of these computations are given in (Cleland et al. 2007; Cleland and Sethupathy 787 2006). Biv shows how spikes generated in Mi cells in response to activation patterns (left 788 side) are transformed into sparser, oscillatory and highly synchronized spike patterns by 789 the interactions with deeper interneuron networks (Escanilla et al. 2010; Mandairon et al. 79