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Mechanisms Underlying Early Odor Preference Learning in Rats

5

Qi Yuan*,1, Amin MD. Shakhawat*, Carolyn W. Harley{,1 *

Biomedical Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada { Department of Psychology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada 1 Corresponding authors: Tel.: 17097772399 (Yuan); 17098647974 (Harley); Fax: 17097777010 (Yuan); 17098642430 (Harley), e-mail address: [email protected]; [email protected]

Abstract Early odor preference training in rat pups produces behavioral preferences that last from hours to lifetimes. Here, we discuss the molecular and circuitry changes we have observed in the olfactory bulb (OB) and in the anterior piriform cortex (aPC) following odor training. For normal preference learning, both structures are necessary, but learned behavior can be initiated by initiating local circuit change in either structure. Our evidence relates dynamic molecular and circuit changes to memory duration and storage localization. Results using this developmental model are consistent with biological memory theories implicating N-methyl-D-aspartate (NMDA) receptors and b-adrenoceptors, and their associated cascades, in memory induction and consolidation. Finally, our examination of the odor preference model reveals a primary role for increases in a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptor synaptic strength, and in network strength, in the creation and maintenance of preference memory in both olfactory structures.

Keywords learning, memory, odor, long-term potentiation, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, N-methyl-D-aspartate receptor, norepinephrine, adrenoceptor, olfactory bulb, anterior piriform cortex

1 INTRODUCTION Early odor preference learning is a form of rapid classical conditioning that has proved to be a useful model for identifying circuit, cellular, and molecular learning mechanisms (Sullivan and Wilson, 2003; Wilson and Sullivan, 1994). Newborn rats Progress in Brain Research, Volume 208, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63350-7.00005-X © 2014 Elsevier B.V. All rights reserved.

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can be conditioned to novel odors using stimulation that mimics the stimulation received during maternal care. A variety of stimuli have been used as the unconditioned stimulus (UCS) to induce conditioned responses to novel odors (conditioned stimulus, CS) in neonates, including the nesting environment (Alberts and May, 1984; Galef and Kaner, 1980), milk presentation (Johanson and Hall, 1979, 1982; Johanson and Teicher, 1980), stroking or tactile stimulation (McLean et al., 1993; Moore and Power, 1992; Pedersen et al., 1982; Sullivan and Hall, 1988; Sullivan and Leon, 1986; Weldon et al., 1991), tail pinch (Sullivan et al., 1986), the odor of maternal saliva (Sullivan et al., 1986), mild foot shock (Camp and Rudy, 1988; Moriceau et al., 2006; Roth and Sullivan, 2001, 2003; Sullivan, 2003), and intracranial brain stimulation (Wilson and Sullivan, 1990). Pups display a variety of conditioned responses to the CS odor (Johanson and Hall, 1982; Sullivan and Hall, 1988; Wilson and Sullivan, 1994) and the CS appears to acquire the ability to enhance ongoing adaptive behaviors such as huddling, independent feeding (Sullivan and Leon, 1986), and nipple attachment (Pedersen et al., 1982). Leon and colleagues (Coopersmith and Leon, 1984; Leon et al., 1977) first showed that following peppermint odor exposure for the first 19 days of life (3 h/day), rat pups demonstrated a behavioral preference for peppermint when tested at postnatal day (PND) 20. However, odor preference could also be induced with briefer pairings, for instance, 10 min/day odor exposure on PND 1–18, coupled with tactile stimulation, induced a clear odor preference on PND 19 (Sullivan and Leon, 1986). Sullivan and colleagues demonstrated the associative nature of early odor preference behavior. Only those pups with concurrent odor and tactile stimulation developed a conditioned approach to the trained odor: CS-only, UCS-only, random CS–UCS presentations, and backward UCS–CS presentations all failed to induce preference for the trained odor (Sullivan et al., 1989a,b). In the first week of life, pups will even learn a preference for an odor associated with tail pinch or mild foot shock (0.5 mA). However, during the second and third postnatal weeks, mild foot shock induces odor aversive responses in pups (Camp and Rudy, 1988; Moriceau et al., 2006; Sullivan et al., 2000a) and stroking loses its effectiveness as a UCS (Woo and Leon, 1987). Pups trained with odor and concurrent tactile stimulation after the first postnatal week (after
the first 10 days) do not develop a preference for the trained odor on PND 19 (Woo and Leon, 1987). These results suggest a sensitive period for the development of early odor preferences. In the majority of experiments reported here, a single 10 min pairing of stroking and odor is given on PND 6. This single trial pairing induces an odor preference lasting only 24 h. Studies with more extended pairings have provided evidence that odor memory for infantile trained odors can be seen in adulthood (Coopersmith and Leon, 1986) and can influence adult sexual behaviors in male rats (Fillion and Blass, 1986). Several early lines of evidence suggested that norepinephrine (NE) plays a major role as the UCS in early odor preference learning. For example, during birth, both the parturient female and her pups experience unusually elevated NE levels (Sperling et al., 1984), and it was hypothesized that this heightened catecholamine activity

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mechanistically facilitates early odor conditioning (Sulyok, 1989). As will be discussed, Sullivan and colleagues have shown that locus coeruleus (LC)-induced NE release is both necessary and sufficient for infant olfactory learning (Sullivan et al., 2000b). The sensitive period for the tactile UCS in odor preference learning appears to be governed by the development of a2-adrenoreceptor autoinhibition in the LC, which alters its firing pattern to stroking (Kimura and Nakamura, 1987; Nakamura and Sakaguchi, 1990; Nakamura et al., 1987). Our group has focused on identifying and describing the biological mechanisms that support odor preference learning. The evidence reveals dynamic molecular and cellular changes taking place in olfactory structures during memory initiation, consolidation, and storage. The aim of this chapter is to summarize current understanding of the molecular and circuitry changes taking place in the olfactory bulb (OB) and the anterior piriform cortex (aPC) in association with early odor preference learning. Although both structures are necessary for normal preference learning, learned behavior can be modified by local circuit change in either structure via pharmacological manipulations.

2 OLFACTORY BULB Previous investigations of odor preference learning in the rat pup led to the hypothesis that the OB itself is the site of olfactory preference learning and memory (Sullivan et al., 2000b; Yuan et al., 2003b). Here, we review evidence for this hypothesis and then describe more recent experiments that extend our understanding of the role of the OB in supporting odor preference learning and memory. It has been appreciated for sometime that direct infusion of drugs that antagonize neural plasticity into the OB prevents odor preference learning without altering spontaneous odor responses (Lethbridge et al., 2012; Sullivan et al., 1989a; Wilson and Sullivan, 1994). More tellingly, odor preference learning itself can be induced by local OB infusion of plasticity promoters paired with odor (Christie-Fougere et al., 2009; Grimes et al., 2012; Lethbridge et al., 2012; Sullivan et al., 2000b). These effects taken together led Sullivan to suggest that the OB was both necessary and sufficient for odor preference learning (Sullivan et al., 2000b). Early investigators reported local changes in bulbar metabolism and electrophysiology following odor preference training and correlating with odor preference memory. The evidence for changes in bulbar circuitry in support of odor preference learning will be reviewed in subsequent sections.

2.1 Organization of the OB (Simplified Anatomy) Our hypothesis about the OB underpinnings of odor preference learning focuses on the role of the mitral cell (MC) (Yuan et al., 2003b). The MCs receive odor input from the olfactory receptor neurons and send the output of OB processing to the rest of the brain through their axons in the lateral olfactory tract (LOT). The output of MCs is

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modified by a large cohort of inhibitory granule cells. Tufted cells with similar circuitry to that of the MCs also output from the OB, but they are not part of our focus. The apical dendrites of the MCs interface with olfactory neuron axons in specialized complexes on the surface of the bulb called glomeruli. Local inhibitory interneurons, referred to as periglomerular cells, modify and modulate coding activity in the glomeruli. There are thought to be
1000 olfactory sensory receptors in the rodent projecting to
2000 glomeruli with relatively little overlap (Buck and Axel, 1991; Mombaerts et al., 1996; Ressler et al., 1993). Odors activate distinct glomerular maps depending on odorant composition (Mombaerts et al., 1996).

2.2 Molecular Underpinnings for Learning in the OB Over the past 20 years, our understanding of molecular process underpinning learning and memory has been accelerated by studies of neural circuits within both invertebrate and vertebrate species. The review in the subsequent sections will focus on cellular and molecular mechanisms underlying early odor preference learning. Specifically, we will emphasize plasticity-associated neural substrates of the rat neonate’s odor associative memory.

2.2.1 LC–Noradrenergic Input A unique early pattern of signaling from the LC to the OB is thought to provide the UCS for normal early odor preference learning. The LC is the major source of NE in the OB. The LC projects about 40% of its fibers to the OB (Shipley et al., 1985) and has a heightened sensitivity to sensory input during early developmental stages (Nakamura and Sakaguchi, 1990; Nakamura et al., 1987). The densest projection of these fibers is known to be in the internal plexiform and granule cell layers. Comparatively, less dense projections were found in the external plexiform layer, and projections were also sparse in the glomerular layer (McLean et al., 1989). LC fibers are present (McLean and Shipley, 1991) and functional (Wilson and Leon, 1988a) in the rat neonatal bulb in the first week of rodent life. Nakamura et al. (1987) has shown that tactile stimulation, including stroking, tail pinch, and air puff, evoked clear and prolonged responses from LC neurons as early as PND 1. NE released from the LC terminals following tactile stimulation (Nakamura et al., 1987; Rangel and Leon, 1995) is required for acquisition of conditioned odor preference. Pharmacological blocking of noradrenergic b-adrenoreceptors in the bulb prevents odor learning (Sullivan et al., 1989b, 1991, 2000b). Odor preference can also be rapidly acquired by pairing odor stimulation with pharmacological activation of b-adreneroreceptors (Harley et al., 2006; Langdon et al., 1997; Lethbridge et al., 2012; Sullivan et al., 1989b, 2000b; Yuan et al., 2003b) or by direct stimulation of LC (Sullivan et al., 2000b). Bulbar-specific manipulations (see Sullivan et al., 2000b) provide the evidence for the hypothesis that bulbar NE is both necessary and sufficient for early odor preference learning. Several neonatal properties of the LC support the learning-associated plasticity of the bulb. First, inhibitory a2 noradrenergic autoreceptors are not functional at this age

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(Nakamura and Sakaguchi, 1990; Nakamura et al., 1987; Winzer-Serhan and Leslie, 1999), which increases the response duration of LC from a few milliseconds to 10–30 s in rat pups compared to adult rats (Nakamura et al., 1987). Second, immature LC neurons in the newborn pups are responsive to a wide range of stimuli and are more electrically coupled compared to the mature LC in adult rats (Christie et al., 1989; Nakamura et al., 1987). The immature LC physiology increases the probability that LC neurons will be synchronously activated for extended periods by nonnoxious UCSs. Odor plus tactile stimulation induces a marked increase of NE in the OB in pups in the first week, which is not seen to odor alone, and is greater than that seen to tactile stimulation alone. NE levels are lower to combined stimulation by 10 days of age (Rangel and Leon, 1995). NE in neonates also reduces MC habituation to repetitive odor presentations during associative training (Wilson and Sullivan, 1992). These early features of LC output support the ability of pups to efficiently make odor–UCS associations during the critical period (Moriceau and Sullivan, 2004). Outside of the critical period, Moriceau and Sullivan (2004) have also induced odor preference conditioning by mimicking the pattern of LC activation that occurs in the critical period. Direct infusion of a b-adrenoceptor agonist in the OB also produces postcritical period odor preference learning. This argues that the bulbar plasticity mechanisms recruited by NE release in the neonate are present in the more mature OB. There are multiple NE receptors in the bulb, and the mechanisms by which NE promotes learning-induced plasticity are likely to be multifaceted. Although LC fibers project differentially into the different layers of the bulb, adrenoceptors are expressed in all layers of the bulb. Both MCs and granule cells express a-adrenoceptors, including a1 and a2 subtypes (Day et al., 1997; Hayar et al., 2001; McCune et al., 1993; Nai et al., 2010; Pieribone et al., 1994; Winzer-Serhan and Leslie, 1999; Winzer-Serhan et al., 1997a,b). Both b1- and b2-adrenoceptors have also been reported, using radioautographic techniques, in the granule cell, internal plexiform, and glomerular layers, with only b2-adrenoceptors occurring in the external plexiform layer (Woo and Leon, 1995). Later, antibody localization experiments demonstrated that b1-adrenoceptors are most conspicuous on MCs and periglomerular cells and have only a minor distribution on granule cells (Yuan et al., 2003b). We turn now to a consideration of the evidence for participation of multiple bulbar adrenoceptors in the initiation and support of early odor preference learning. It is likely that release of NE from LC terminals in the OB supports odor preference conditioning via all of the receptor subtypes discussed below.

2.2.1.1 b-Adrenoceptors

Early pharmacological evidence demonstrated that systemic blocking of b-adrenoceptors with propranolol in the rat pup prevented preweanling olfactory learning (Sullivan et al., 1989b). Subsequent studies showed the antagonist effect occurs even when administration of the b-adrenoceptor blocker is restricted to the OB (Sullivan et al., 2000b). Critically, when odor is paired with the b-adrenoceptor agonist isoproterenol (a nonspecific b1 and b2 agonist that crosses the blood–brain barrier in rat

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pups, but not adults) at 2 mg/kg, an odor preference is produced (Sullivan et al., 2000b). A later study using separate systemic b1 and b2 agonist administration suggested that odor preference learning induction is specific to b1-adrenoceptor activation (Harley et al., 2006). b-Adrenoceptor activation as a UCS exhibits an inverted U-shaped dose– response curve. A moderate dose of isoproterenol (2 mg/kg) paired with a novel odor induces preference; however, odor paired with a lower dose (1 mg/kg) or higher doses (4–6 mg/kg) does not (Langdon et al., 1997; Sullivan et al., 1989b; Yuan et al., 2003b). Again this effect can also be seen with selective b1 agonist administration (Harley et al., 2006). Consistent with mediation of the stroking UCS through LC release of NE, the pairing of a low dose of isoproterenol with an ineffective stroking stimulus synergistically produces learning, while pairing stroking with the optimal isoproterenol UCS prevents learning presumably due to excessive stimulation (Sullivan et al., 1991). These effects suggest that the two inputs are summating. Since isoproterenol infusion directly in the OB paired with a novel odor also produces odor preference learning, it is likely that LC-mediated NE release and isoproterenol effects are converging on OB b1-adrenoceptors that provide the critical UCS.

2.2.1.2 a-Adrenoceptors

The role of b-adrenoreceptors in early odor preference learning has been extensively investigated (Langdon et al., 1997; Sullivan and Leon, 1986; Sullivan et al., 1989b, 1991, 2000b; Yuan et al., 2003b); however, the role of a-adrenoreceptors in this learning paradigm has only recently received attention. Harley et al. (2006) found that systemic injection of the a1-adrenoceptor agonist, phenelyephrine, paired with odor, also produced odor preference learning and doses exhibited an inverted U-curve relationship to learning. There were no learning effects with a systemic a2 agonist for the doses explored. However, in a recent study, we induced rat pup odor preference learning by infusing 500 mM clonidine (a2-adrenoceptor agonist) directly into the OB (Shakhawat et al., 2012). Lower concentrations were ineffective. To control for a potential effect of clonidine on the a1-adrenoceptor, a cocktail of prazosin (a1-adrenoceptor antagonist) and clonidine was also infused. The coinfusion group still showed a significant learning effect compared to controls. We were able to prevent odor learning induced by pairing odor and a mild shock with a bulbar infusion of the a2-adrenoceptor antagonist yohimbine. These findings provide support for the hypothesis that a2-adrenoceptor activation can also act as a UCS for odor preference learning. Coapplication of a suboptimal dose (50 mM) of clonidine (an a2 agonist) synergized with a lower 1.5 mg/kg dose of isoproterenol, suggesting reinforcing support of odor preference learning through these two receptor subtypes in the OB.

2.2.2 Serotoninergic Support Mclean et al. (1993) initially showed that depleting serotonin locally on PND 1 from axons innervating the OB prevented the later acquisition of conditioned odor learning. It was later found that this effect of 5-HT depletion could be overcome by

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increasing the dose of isoproterenol used as the UCS (Langdon et al., 1997). The receptor mediating serotonin support of odor preference learning was found to be the 5HT2 receptor as learning could be prevented by systemic injections of the 5HT2A/2C antagonist ritanserin (McLean et al., 1996). Learning could be induced with stroking as the UCS in 5HT-depleted pups, if there was a prior subcutaneous injection of a 5HT2A/2C agonist (McLean et al., 1996). In an extensive dose–response study, it was not possible to induce learning by pairing novel odor and the 5HT2A/2C agonist alone (Price et al., 1998). This pattern of results suggested that serotonin acts to support normal b-adrenoceptor mediation of odor preference learning. We investigated the colocalization of b1-adrenoceptors and 5HT2 in the OB and found both occurred on MCs (Yuan et al., 2003b). Other literature suggested that the 5HT2 receptor synergistically enhances the cAMP modulation initiated by b1-adrenoceptors (Morin et al., 1992) in neocortex. As will be described, this also occurs in the OB.

2.2.3 Cyclic Adenosine Monophosphate A role for cyclic adenosine monophosphate (cAMP) as a critical intracellular signal in the initiation of associative learning has been demonstrated in a wide variety of species (Aplysia (Brunelli et al., 1976; Pittenger and Kandel, 2003); Drosophila (Byers et al., 1981; Shotwell, 1983; Yin and Tully, 1996); Rodents (Bourtchuladze et al., 1994; Cui et al., 2007; Yuan et al., 2003b)). Since the groundbreaking discoveries in Aplysia and Drosophila, a growing body of evidence supports the hypothesis that the cAMP/protein kinase A/cAMP response element binding protein (cAMP/PKA/CREB) cascade might be a universal mechanism underlying learning and memory. While there is indirect evidence for such an assertion in mammalian models, direct evidence of cAMP’s role in a defined mammalian learning circuit is scarce (Alberini, 1999). We have proposed a classical conditioning model of odor preference learning in rat pups, in which MC cAMP increases mediate the UCS (Yuan et al., 2003b). Given the critical role of bulbar b-adrenoceptors acting via b1-adrenoreceptor activation in inducing odor preference memory when paired with a novel odor, it was logical to propose a causal role for cAMP in driving odor preference memory associations (McLean et al., 2005). The first direct evidence consistent with the hypothesis was the discovery that colocalized b1-adrenoceptors and 5-HT2A receptors on MCs were both required for the increase in MC cAMP staining, normally seen 10 min after the pairing of effective UCS and odor input. Depletion of 5-HT prevented both learning with typically effective UCS and the cAMP increase in MCs normally observed (Yuan et al., 2003b). A causal role for the cAMP increases in odor preference learning was demonstrated by manipulating cAMP levels with a phosphodiesterase IV inhibitor, cilomilast. The inhibitor converted a low learning-ineffective dose of isoproterenol (1 mg/kg) into an effective UCS (McLean et al., 2005). In a follow-up experiment, cilomilast restored learning in pups with 5-HT-depleted bulbs when paired with 2 mg/kg isoproterenol, a condition that had previously been shown ineffective in producing learning (McLean et al., 2009).

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The detailed nature of an optimal cAMP learning signal has not been described in most animal learning models. That such a pattern exists is suggested by work in slime mold where cAMP serves as an aggregation signal and only oscillatory or pulsating patterns created by calcium/cAMP feedback interactions are effective (Goldbeter, 2006; Goldbeter et al., 1988, 2000). The likelihood that cAMP signal patterning might be critical in early odor preference learning was suggested by the observation that higher doses of isoproterenol, which are effective in increasing cAMP in the OB, do not produce learning when paired with odor (Yuan et al., 2003b). Cui et al. (2007) investigated bulbar cAMP temporal patterning in relation to rat pup odor preference learning. They found that cAMP peaked 10 min following training in normal effective conditioning, that is to say, levels first increased to a peak at 10 min and then decreased. These changes were later shown immunohistochemically to be occurring in MCs (Cui et al., 2007; Yuan et al., 2003b). With high doses of isoproterenol as the UCS, there was only a sustained rising increase in cAMP levels over the 20-min period following training. The normally effective isoproterenol dose produced a similar rising pattern when given alone, but when combined with novel odor input, the 10min peak elevation in cAMP followed by a decrease was observed (Cui et al., 2007). An ineffective isoproterenol dose that produced no learning when paired with odor did not elevate cAMP in the bulb unless combined with a phosphodiesterase inhibitor. When so combined, the pulsatile cAMP pattern emerged (Cui et al., 2007). Thus, an interaction between intracellular signals generated by novel odor input and by the UCS is required for learning and induces a pulsatile modulation of cAMP in MCs. The natural stimulus of stroking paired with odor also produces the 10-min cAMP peak followed by a decrease pattern. Unpaired odor or stroking alone does induce natural pulsatile patterns, but their timing is delayed relative to those seen with pairing (Cui et al., 2007). The causal role of cAMP in rat pup early odor preference requires specific spatiotemporal patterning in the OB.

2.2.4 Protein Kinase A PKA is known to play a highly conserved key role in long-term memory (LTM) formation both in vertebrates (Abel and Nguyen, 2008) and invertebrates (Barco et al., 2006). PKA is activated as a primary action of cAMP (Dell’Acqua and Scott, 1997; Taylor et al., 1990), and in turn, catalytic subunits of PKA are known to phosphorylate learning-related downstream substrates such as serine 845 of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluA1 subunit (Ahn and Choe, 2009; Banke et al., 2000) and serine 133 of the transcription factor CREB (Ahn and Choe, 2009; Delghandi et al., 2005). Since cAMP is causal in odor preference learning (McLean et al., 2005, 2009), and CREB phosphorylation is engaged by early odor preference learning (McLean et al., 1999; Yuan et al., 2003b), it is not surprising that a recent series of experiments (Grimes et al., 2012) have demonstrated a causal role for PKA activation in rat odor preference learning. As predicted from the pattern of cAMP increases with odor preference conditioning, PKA activation is maximal 10 min following training during the cAMP peak. Intrabulbar infusions of the PKA antagonist (Rp-cAMPs) prevent normal odor

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preference learning. CREB phosphorylation does not occur, which is again consistent with a causal role for the cAMP/PKA/CREB cascade. The PKA agonist, Sp-cAMPs, directly infused into the OB, together with novel odor presentation itself, acts as a UCS and creates a normal 24-h odor preference memory. Infusions of Sp-cAMPs doses higher than the minimally effective concentration do not, however, produce an inverted U-curve. Instead, higher concentrations result in more enduring odor preference memory, with a preference still expressed 48 and 72 h after training, in contrast to the 24-h memory produced by normal single trial training. Consistent with other literature suggesting that the cAMP/PKA/CREB cascade is selectively involved in late-phase synaptic plasticity and long-term, rather than short-term, memory (Alberini et al., 1995; Bailey et al., 1996; Huang et al., 1994; Nguyen and Kandel, 1996, 1997; Nguyen et al., 1994), rat pups trained with the PKA activator Sp-cAMP as a UCS did not show short-term preference memory at 3 h. The first indication of a preference memory was seen 5 h following training. Intermediate-term memory (ITM) has also shown to be PKA dependent in invertebrates (Sutton and Carew, 2000; Sutton et al., 2001). This suggests that PKA plays a causal role in intermediate-term and longterm preference memory in the rat pup odor preference model, but not in short-term memory (STM). These data argue strongly for separate and parallel cellular memory mechanisms underlying normal learning (Grimes et al., 2012).

2.2.5 Phosphatase Although it is well recognized that kinases play a key role in memory, phosphatases have been less extensively studied. In our odor preference learning model (Yuan et al., 2003b), both protein phosphase 1 and calcineurin (protein phosphatase 2B) are suggested to mediate the dephosphorylation of CREB. They, in turn, can be inhibited by the cAMP-sensitive inhibitor 1 (Fig. 1). The extension of memory induced by supra PKA activation may relate to a longer activation of inhibitor 1 and a longer duration of CREB phosphorylation. This remains to be investigated. However, the calcium-dependent phosphatase, calcineurin, shown to be colocalized with PKA at postsynaptic densities (Coghlan et al., 1995; Yakel, 1997), has been manipulated in the odor preference learning model. Calcineurin dehosphorylates phosphodiesterases and adenylate cyclases as well as CREB (Lin et al., 2003a,b; Snyder et al., 2003; Yang et al., 2004). All of these dephosphorylating actions limit the duration of associative intracellular events in the one-trial odor preference training model. We proposed earlier (Yuan et al., 2003b) that higher doses of isoproterenol and their associated sustained cAMP levels might favor phosphastase activation, altering the balance of phosphorylating and dephosphorylating events in MCs to block associative learning. Thus, stronger dephosphorylation activity could underlie the dose-dependent inverted U-curve associated with strong UCS activation. Calcineurin inhibition localized to the OB using the calcineurin inhibitor FK506 does not prevent normal 24-h odor preference memory (Christie-Fougere et al., 2009). Rather, it facilitates such learning when paired with a suboptimal dose of

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FIGURE 1 Proposed model for intracellular pathways in the olfactory bulb activated by b1-adrenoceptors and 5-HT2 receptors during early odor preference learning in rats. b1-Adrenoceptors mediate the unconditioned stimulus (UCS) via either tactile stimulation (activating locus coeruleus (LC)-mediated norepinephrine release) or a b-adrenoceptor agonist and trigger the cAMP cascade. The conditioned stimulus (CS) is provided by odors that stimulate glutamate receptors on mitral cells and results in calcium influx through NMDARs and L-type calcium channels. When UCS and CS are paired, the intracellular pathways induce phosphorylation of CREB and 24 h memory. Serotonergic input from raphe nuclei facilitates b-adrenoceptormediated cAMP productions and learning. Reproduced from Yuan et al. (2003b), with permission from Cold Spring Harbor Laboratory Press.

isoproterenol as the UCS. FK506 cannot itself act as a UCS, however. With normal training parameters, blocking the calcium-dependent phosphatase extends odor preference memory, as our model suggests, with preferences expressed up to 96 h after training using the lowest facilitating dose of FK506. CREB phosphorylation duration was also extended by this treatment, suggesting the hypothesis that the duration of CREB phosphorylation positively modulates memory duration. If the calcineurin antagonist is infused when 6 mg/kg of isoproterenol is used as the UCS, a dose that normally does not produce odor preference, memory is seen. This is consistent with the prediction that phosphatases play a critical role in the inverted U-curve for the UCS as was the evidence that direct PKA activation alone produces conditioning without a dose-dependent inverted U-curve.

2.2.6 CREB CREB activation was first described in Aplysia and Drosophila as required for the conversion of STM to LTM (Brunelli et al., 1976; Byers et al., 1981; Dudai et al., 1983). Later, molecular manipulation of CREB function in mammals confirmed its hypothesized role in LTM formation and memory-related synaptic plasticity

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(Pittenger et al., 2002; Tully et al., 2003). The majority of paradigms have examined aversively motivated learning, for example, olfactory avoidance conditioning in fruit flies (Tully et al., 2003; Yin and Tully, 1996; Yin et al., 1995), defensive withdrawal conditioning in Aplysia (Abel and Kandel, 1998; Bartsch et al., 1998; Michael et al., 1998), and avoidance conditioning and spatial escape learning in rodents (Silva et al., 1998). It has been referred to in reviews as “the memory gene” (Yin and Tully, 1996). If CREB is critical in olfactory preference learning, consistent with the involvement already described of cAMP and PKA in the appetitive olfactory preference learning, then CREB phosphorylation should be an early step in the development of 24 h memory. Using Western blot analysis, we observed a significant increase in phosphorylated CREB (pCREB) levels in the OB 10 min following olfactory conditioning training (odor þ stroking), which reliably induces 24-h odor preference memory in pups (McLean et al., 1999). Pups that are given nonassociative training only (odor only or stroking only) fail to show increased pCREB. In trained pups, CREB phosphorylation declines, but is still elevated 30 min after conditioning, and is not seen at 60 min. The increased phosphorylation is also not seen immediately after training. This suggests a temporally focused and restricted period of CREB activation. Spatially, increased pCREB is localized to MC nuclei within the dorsolateral quadrant of the bulb of pups undergoing peppermint odor stroke pairing (McLean et al., 1999). This is the region of maximal peppermint activation metabolically (Johnson et al., 1995). This selective recruitment of MCs is consistent with the general theory we presented. Significant differences were absent among nonlearning groups (Naı¨ve, odor only, or stroke only) or among any training groups in the granule or periglomerular cells of the dorsolateral region (McLean et al., 1999). Using 2 mg/kg of isoproterenol paired with peppermint, instead of tactile stimulation as the UCS, we replicated the finding that pCREB is increased with odor preference learning (Yuan et al., 2000). Using isoproterenol permits an examination of the inverted U-curve associated with the UCS in odor preference learning. A 6 mg/kg concentration of isoproterenol failed to produce learning and did not lead to an increase in CREB phosphorylation. This occurred despite the elevated cAMP levels that normally follow 6 mg/kg injections of isoproterenol when paired with odor (see earlier discussion in Section 2.2.3). Learning that was prevented by selective serotonin depletion in the OB could be restored by the higher 6 mg/kg dose of isoproterenol. In that instance, pCREB levels were also significantly elevated, while they were not elevated when only 2 mg/kg of isoproterenol was paired with odor in 5-HT-depleted pups. The occurrence of learning, not the concentration of the b-adrenoceptor activator employed, was the controlling variable for CREB phosphorylation 10 min after training. This result is consistent with CREB as a “memory gene” in this model. Finally, the causal role of CREB and pCREB in early odor preference learning has been directly tested by injecting a Herpes simplex virus expressing either CREB (HSV-CREB) or a dominant-negative mutant CREB (HSV-mCREB) bilaterally into the OB (Yuan et al., 2003a). HSV-LacZ expressing Escherichia coli b-galactosidase

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was used to determine whether the virus injection itself would affect odor preference learning and the extent of spread of the viral infusion. Injection of HSV-mCREB prior to training prevented normal early odor preference learning; HSV-LacZ-injected pups showed normal preference to the conditioned odor after odor þ stroking training. Unexpectedly, increased CREB levels created by prior injection of HSV-CREB also interfered with odor þ strokinginduced learning. Both CREB levels and CREB phosphorylation were significantly higher in the HSV-CREB group than in the learning successful HSV-LacZ group (Yuan et al., 2003a). These data argue that there is an inverted U-curve effect for the amount of CREB phosphorylation itself. It is unlikely that such exaggerated CREB levels would occur in normal rat pups, however. The use of isoproterenol permitted us to investigate the role of UCS strength in the presence of additional CREB support or of anomalous mutant CREB. The control group (HSV-LacZ) showed the typical inverted U-curve learning effect with varying concentrations of isoproterenol, increasing CREB levels by an HSV-CREB injection shifted the dose–learning curve for isoproterenol to the left, such that an originally ineffective learning dose, 1 mg/kg isoproterenol, now induced significant learning when paired with odor. Expression of mutant CREB (the HSV-mCREB injection) shifted the dose–learning curve for isoproterenol to the right, with a higher dose of isoproterenol now producing learning (Yuan et al., 2003a). These data suggest that with more CREB available lower levels of PKA activity suffice to reach a learning threshold for CREB phosphorylation, while with ineffective CREB occupying some portion of the phosphorylation mechanisms, a higher level of PKA activation is required. Taken together, these results argue that an optimal amount of CREB phosphorylation 10 min post-training is the gateway for initiating 24-h memory in the early odor preference learning model.

2.2.7 Protein Synthesis Temporally, and mechanistically, memories have been characterized as having multiple phases, typically, short-term memory (STM), intermediate-term memory (ITM) and long-term memory (LTM) (Davis and Squire, 1984; McGaugh, 2000; Rosenzweig et al., 1993). In addition to the temporal differences among these memories, there are differences in their dependence on protein synthesis. STM does not require protein synthesis. ITM depends on translation but does not require mRNA transcription. LTM requires both translation of mRNA and transcription of mRNA (Castellucci et al., 1989; Davis and Squire, 1984; McGaugh, 2000; Montarolo et al., 1986; Pinsker et al., 1973; Rosenzweig et al., 1993; Sutton and Carew, 2000; Sutton et al., 2001). In the rat pup odor preference learning model, these three memory types have also been characterized (Grimes et al., 2011). STM lasts up to 3 h after training. ITM is currently defined as occurring 5 h after training and LTM is seen at 24 h. As predicted from studies in invertebrates, rat pup odor preference ITM is disrupted by a translation inhibitor (anisomycin) infused into the OB, but not by a transcription inhibitor (actinomycin). Neither inhibitor interferes with STM. LTM is prevented by either inhibitor (Grimes et al., 2011). The apparent time course of learning-related

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critical protein synthesis for 24 h memory is within a 1-h window after training. Inhibiting protein synthesis 3 h after training has no effect on 24 h memory. The rat pup olfactory preference learning model captures the protein synthesis-dependent features of memory previously described in invertebrate and vertebrate models (Castellucci et al., 1989; Davis and Squire, 1984; McGaugh, 2000; Montarolo et al., 1986; Pinsker et al., 1973; Rosenzweig et al., 1993; Sutton and Carew, 2000; Sutton et al., 2001). It may be the first mammalian model to clearly demonstrate ITM. The delineation of a time window for associative learning-critical protein synthesis helps to direct the identification of proteins underlying the circuit remodeling required for memory.

2.2.8 AMPA Receptors Glutamate released from the olfactory nerve (ON) activates MCs via NMDA receptors (NMDARs) and AMPA receptor (AMPAR) (Aroniadou-Anderjaska et al., 1997). Temporary and/or permanent modification(s) of the neuronal response to this excitatory transmitter is a putative mechanism of olfactory learning. In this, and the following section (2.2.9), we discuss the role of AMPAR and NMDAR in our cAMP/PKA/pCREB-dependent appetitive learning model. Activity-dependent trafficking of AMPAR in the synapse is hypothesized to support the formation of memories (Malinow and Malenka, 2002). Considerable evidence suggests that reversible phosphorylation of the AMPAR subunit GluA1 contributes to both long-term potentiation (LTP) and long-term depression (LTD) (Jensen et al., 2003; Lee et al., 2003, 2010; Meng et al., 2003; Zamanillo et al., 1999). The trafficking and function of AMPAR are regulated by two phosphorylation sites, Ser845 (a PKA site) and Ser831 (a PKC/CaMKII site), situated in the GluA1 subunit (Banke et al., 2000; Barria et al., 1997; Lee et al., 2003, 2010). It has been shown that b-adrenoceptor activation leading to PKA-dependent Ser845 phosphorylation facilitates the synaptic delivery of GluA1-containing AMPARs and LTP and lowers the threshold for the formation of an aversive emotional memory (Hu et al., 2007). Since early odor preference memory appears to be mediated by the cAMP/PKA/ CREB cascade (Fig. 1), the model permits direct testing of the hypothesis that PKAmediated GluA1 receptor phosphorylation on serine 845, and/or changes in the distribution or levels of the GluA1-containing AMPAR, is initiated with appetitive learning and supports memory. Cui et al. (2011) found a significant increase in phosphorylated GluA1 (pGluA1) in the OB of neonates following pairing of odor with the optimal isoproterenol dose (2 mg/kg), a conditioning paradigm that leads to odor preference learning. Maximum increases of pGluA1 levels were observed at 10 min after conditioning. An elevated level of pGluA1 was observed for up to 1 h post-conditioning and then returned to basal levels. Interestingly, the highest level of pCREB expression is also found 10 min after conditioning (McLean et al., 1999; Yuan et al., 2000). Although novel odor only exposure leads to an intermediate level of pGluA1 expression, isoproterenol (2 mg/kg) alone fails to induce any change in the expression level of pGluA1 in

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the bulb. This pattern of PKA-mediated phosphorylation of the GluA1 subunit is consistent with our model in which the pairing of odor and b-adrenoceptor activation initiates learning through the activation of the cAMP/PKA/CREB cascade. Early increases in AMPAR currents may be supported by phosphorylation. These results further demonstrate that the association of CS and UCS is a prerequisite for S845 phosphorylation of the GluA1 subunit. As expected, the total GluA1 expression level is unchanged in the learning group 10 min after training. Total GluA1 expression was also unchanged at 24 h. The AMPAR hypothesis does not necessarily predict any change in total GluA1 levels, but rather a change in subcellular distribution. This was examined by using a synaptoneurosomal preparation from trained and control rat pups (Cui et al., 2011). As predicted, there was a significantly higher GluA1 distribution in the synaptoneurosomes of rat pups 24 h after training as compared to an odor only subgroup at the same time point or to naı¨ve pups. This finding is clear support of the increased AMPAR membrane insertion model of circuit modification underlying memory. Cui et al. next used immunocytohistochemistry to further follow, and localize, the increases in the GluA1-containing AMPAR. Increases in immunoreactivity in the glomerular regions were seen both at 3 and 24 h after training. These increases are consistent with the prediction of an increase in ON input strength. Both STM (3 h) and LTM (24 h) appear related to AMPAR insertion. The increase in GluA1-containing AMPAR staining was not seen at either 10 min or 48 h after training. As memory expression, itself, is not seen 48 h after a single training trial, this is consistent with the predictions of the AMPAR hypothesis. Reversible modifications like phosphorylation may provide memory support at the earliest time points. The failure of PKA blockade to prevent STM and of PKA activation to initiate STM suggests that other phosphorylation mechanisms must be involved in early memory. To test the causal role of AMPAR insertion in 24-h odor preference memory, Cui et al. used bulbar infusion of a GluA1 interference peptide (Tat-GluA1CT). Twenty-four hour odor preference memory was blocked following antagonist infusion confirming the predicted requirement of AMPAR insertion in LTM.

2.2.9 NMDA Receptors NMDARs respond to glutamate activation, if and only if the membrane in which they are embedded is concurrently depolarized. This feature gives them a special role as an associative neural plasticity mechanism, the “Hebbian coincidence detector” (Bear and Malenka, 1994; Malenka and Bear, 2004). In our original model, we suggested that calcium influx via NMDARs would interact with calcium-sensitive adenylate cyclases to produce the associative learning signal (Yuan et al., 2003b; see Fig. 1), a mechanism first demonstrated for invertebrate learning by Yovell and Abrams (1992). The first study of the role of NMDARs in early odor preference learning used systemic injection to block NMDARs and reported that both the preference for and the enhanced metabolic 2-DG response to the trained odor were suppressed (Lincoln et al., 1988), as predicted by our model. A later investigation, using

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MK-801, confirmed the learning disruption and found that injections immediately posttraining but not 30 or 60 min posttraining had the same disruptive effect (Weldon et al., 1997). This is consistent with a role for NMDARs in the initiation, but not the longer-term support, of learning. More recently, we have examined the effect of intrabulbar infusions of the NMDAR antagonist D-APV (500 mM) (Lethbridge et al., 2012). Interestingly, central infusion enhances MC pCREB levels, consistent with a release of MCs from granule cell inhibition (Schoppa et al., 1998; Wilson et al., 1996). However, infusions directed at the mid-lateral region of the OB, where the peppermint representation area at the glomerular level is located (Johnson and Leon, 1996; Johnson et al., 1995), attenuate pCREB expression in MCs and prevent odor preference learning. This result helps us to draw the conclusion that MC NMDARs responding to the ON input are critical for normal learning. Consistent with our pharmacological localization of the critical site for NMDAR support of odor preference learning, we found an increase in the phosphorylation of the mid-lateral glomerular NMDARs 5 min following odor preference training, the earliest time point examined. Phosphorylation occurred on the PKA-sensitive Serine897 site of the GluN1 subunit. Subsequent Western blot analyses of the GluN1 subunit revealed a significant downregulation of this subunit 3 h post-training. This downregulation suggests that LTP plasticity may be reduced at the 3-h time point. The levels of the GluN1 subunit returned to baseline by 24 h; however, the plasticity-implicated GluN2B (Quinlan et al., 2004) subunit was downregulated at the 24-h time point. Whether these changes support memory stability remains to be assessed.

2.2.10 L-type Calcium Channels L-type calcium channels (LTCCs), a long-opening high-voltage-gated calcium channel, are known to play an important role in triggering intracellular cascades related to synaptic plasticity (Deisseroth et al., 1998; Mermelstein et al., 2000) and in Hebbian synaptic plasticity at glutamatergic synapses (Bauer et al., 2002; Grover and Teyler, 1990, 1992; Weisskopf et al., 1999). We have investigated the potential contribution of these channels to early odor preference learning (Jerome et al., 2012). The LTCC blocker nimodipine coinfused with isoproterenol during odor conditioning prevents odor preference memory. The LTCC activator BayK-8644 rescues isoproterenolinduced learning from NMDAR blockade. This suggests that calcium entry together with b-adrenoreceptor activation is required for odor preference learning and that a proximal route of significant calcium entry in MCs may occur through LTCCs. The LTCC activator BayK-8644 is not itself sufficient to act as a UCS but appears to provide the requisite calcium priming originally proposed in our associative model (Fig. 1). Consistent with a role in MC calcium entry, immunohistochemistry confirms a dense staining of the Cav1.2 subtype of LTCCs in the external plexiform layer in association with the shafts of MC apical dendrites (Jerome et al., 2012). Recently, apical dendritic LTCCs have been shown to be critical for human neuronal

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spike-timing-dependent synaptic plasticity (Verhoog et al., 2013). The present data demonstrate that LTCC activation can overcome even NMDAR blockade to initiate memory if coincident with b-adrenoreceptor activation (Jerome et al., 2012). A role for noradrenergic modulation of LTCCs in supporting memory acquisition, including, possibly, human memory acquisition, is suggested by these findings.

2.2.11 Metabotropic Glutamatergic Receptors Activation of the metabotropic glutamatergic receptors mGluR2/3 by the agonist DCG-IV has been shown to serve as powerful learning signal for mouse female mate memory in the accessory OB (Hayashi et al., 1993; Kaba et al., 1994). Metabotropic mGluR2/3 receptors are expressed in both MCs and granule cells in the accessory OB (Ohishi et al., 1993). In the main OB, these receptors are prominent in periglomerular cells but also occur in other cell types such as granule cells (Neki et al., 1996; Ohishi et al., 1993; Petralia et al., 1996). Infusion of DCG-IV, in the main OB, paired with exposure to peppermint odor, induces odor preference learning (Rumsey et al., 2001). If applied together with odor þ stroking, the same DCG-IV dose prevents odor preference learning. This effect is consistent with a convergence of metabotropic and noradrenergic UCS effects. As seen previously, excessively strong UCSs reveal an inverted U-curve relationship with memory. The effects here may relate to disinhibition of MCs, but it needs to be clarified.

2.2.12 GABAergic Periglomerular and granule cells are the two classes of GABAergic interneurons within the OB that refine both the input (periglomerular cells) and the output (granule cells) of MCs. Both of these interneurons participate in GABAA dendrodendritic communication with their target MCs. GABAB receptors on the ON have also been identified as presynaptic inhibitors of ON transmitter release and are likely modulated by periglomerular cells (Aroniadou-Anderjaska et al., 2000; Murphy et al., 2005). Okutani et al. (1999, 2003) first investigated learning and GABAergic mechanisms in the OB using 30 min of shock–odor pairings with PND11 rat pups. Odor aversions were confirmed on PND12. Infusing the GABAA agonist muscimol into the bulb during training prevented aversive odor learning (Okutani et al., 1999). A similar effect was seen with the GABAB agonist baclofen (Okutani et al., 2003). These results argue for a role of both GABAergic receptors in aversive odor learning. The group also examined the effects of the GABAA antagonist bicuculline and the GABAB antagonist saclofen. At low doses, bicuculline paired with odor produced an odor preference, while high doses produced an odor aversion (Okutani et al., 1999). The GABAB antagonist also produced an odor aversion (Okutani et al., 2003). Even when infused without odor pairings, high doses of either the GABAA or GABAB antagonists produced aversions to all odors tested (Okutani et al., 2002, 2003). This suggests a nonassociative effect of strong disinhibition. However, some level of disinhibition was required for normal associative aversive learning. Okutani et al.’s data also suggest that disinhibition plays a role

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in odor preference learning as well as in odor aversion (Okutani et al., 1999). This hypothesis is supported by our recent physiological studies of the mechanisms underlying early odor preference learning in the OB.

2.3 Physiological Mechanisms in the OB In the next sections (2.3.1 and 2.3.2), we discuss electrophysiological and synaptic mechanisms in the OB, which contribute to early odor preference learning. We first focus on the known effects of NE (as a UCS in learning) on the electrophysiological and synaptic properties of the OB, summarizing the acute and long-term effects of adrenergic activation with a particular focus on our work on the MC and its synapses. We then discuss evidence from ex vivo studies (Lethbridge et al., 2012; Yuan and Harley, 2012) that support an enhanced MC excitation model as the underpinning of early odor preference learning (McLean et al., 1999; Yuan et al., 2003b). The physiological data are consistent with enhanced synaptic transmission at the ON to MC synapses and enhanced postsynaptic AMPAR responses.

2.3.1 Electrophysiological and Synaptic Effects of NE Several lines of evidence indicate that olfactory memories are stored as persistent changes in the circuitry of the OB (Brennan and Keverne, 1997; Wilson and Sullivan, 1994). As already described, NE is a key player. Previous studies have shown that NE enhances excitability and the responses to sensory input in the OB MCs through both direct and disinhibitory mechanisms (Ciombor et al., 1999; Hayar et al., 2001; Jahr and Nicoll, 1982; Jiang et al., 1996; Trombley, 1994). The diversity of adrenoceptor subtypes in the OB (Day et al., 1997; Nicholas et al., 1993a,b; Pieribone et al., 1994; Winzer-Serhan and Leslie, 1999; WinzerSerhan et al., 1997a,b; Woo and Leon, 1995; Yuan et al., 2003b) may account for the complexity of NE effects. As discussed in the previous sessions (2.2.1), multiple adrenoceptors (a1, a2, and b1) have unique roles in early odor preference learning. Here, we describe the diverse physiological effects of NE on cells of the OB as mediated by the different types of adrenoceptors and consider the implications for odor learning.

2.3.1.1 a-Adrenoceptors

NE excites MCs directly via a1 receptors, as either NE or the a1 receptor agonist phenylephrine induces an inward current in MCs that is not blocked by a cocktail of synaptic transmission blockers (Hayar et al., 2001). This effect may underlie increased MC spiking responses induced by NE or phenylephrine to weak ON input in vitro (Ciombor et al., 1999), and, at least in part, account for the increased MC responses to weak odor input during LC activation in vivo (Jiang et al., 1996). Interestingly, a1-adrenoceptor activation can also increase OB granule cell excitability (Mouly et al., 1995). Consistent with such an effect, in the accessory OB, a1adrenoceptor activation enhances GABA release onto MCs (Araneda and Firestein, 2006). Increased excitability in both MCs and granule cells mediated by

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a1-adrenoceptors may improve the signal-to-noise ratio by allowing odor-responsive MCs to remain active while silencing spontaneously active cells. Increased depolarization of strongly activated MCs could recruit NMDAR-mediated processes leading to synaptic changes. a2-Adrenoceptor-mediated effects have also been implicated in altering MC responses in ways that relate to olfactory learning. Earlier studies in the turtle OB (Jahr and Nicoll, 1982) and in dissociated rat OB culture (Trombley, 1994; Trombley and Shepherd, 1992) show that NE disinhibits MCs by suppressing granule cell activity. This effect is attributable to a2-adrenoceptor-mediated presynaptic inhibition of granule cells and/or MC dendrites (Trombley, 1992, 1994; Trombley and Shepherd, 1992). In more recent studies, the a2-adrenoceptor agonist, clonidine, decreases granule cell excitability in acute rat slices (Nai et al., 2010) and reduces synaptic transmission from granule cells to MCs (Pandipati et al., 2010). Consequently, a2-adrenoceptor activation releases the odor-encoding MCs from tonic inhibition and promotes OB synchrony at gamma frequencies (Pandipati et al., 2010). The disinhibition of MCs from granule cell GABAergic effects at dendrodendritic granule cell–MC synapses has long been suggested to play a key role in conditioned olfactory learning (Brennan and Keverne, 1997; Okutani et al., 1999; Wilson and Sullivan, 1994). Such an effect would potentiate ON throughput during acquisition. Finally, a2-adrenoceptor activation produces long-term potentiating effects in OB slices from young rats (