Andrea Darby-King

5-HT2 receptor involvement in conditioned olfactory learning in the neonate rat pup.

These experiments addressed the role of 5-HT2 receptors in conditioned olfactory learning. Ritanserin, a 5-HT2A/2C antagonist, was injected subcutaneously into postnatal day (PND) 7 pups before or after conditioned olfactory training to a peppermint odor. When the pups were tested for olfactory preference on PND 8, those injected with ritanserin before training failed to acquire an odor preference whereas those injected after training learned. This suggested that the 5-HT2 receptor is required only in the acquisition of conditioned olfactory learning. Injection of ritanserin directly into the olfactory bulb before training also blocked preference for the peppermint odor. In pups that had depletion of the 5-HT input to the bulb, subcutaneous injection of a 5-HT2A/2C agonist was sufficient to maintain conditioned olfactory learning, confirming the importance of 5-HT in learning.

Neonatal olfactory sensory deprivation decreases BDNF in the olfactory bulb of the rat.

We hypothesized that brain-derived neurotrophic factor (BDNF) may be down-regulated in the olfactory bulb ipsilateral to experimental naris occlusion. Unilateral naris occlusion was performed on rats at postnatal day three (P3). On P10, P30, and P60 olfactory bulbs were weighed and assayed for tyrosine hydroxylase (TH), BDNF, and TrkB by Western blotting to determine the response of BDNF and its cognate receptor, TrkB, both during the acute phase of sensory loss (P10) and longer term. TH levels, which are highly dependent on intact input from the olfactory epithelium, were assayed as a means of determining the success of occlusion in each animal. At P10, BDNF protein expression was variable but most often increased ipsilateral to deprivation. In contrast, by P30 and P60 TH levels were found to be significantly decreased in the ipsilateral bulbs as were the levels of BDNF. TrkB protein levels changed little relative to the control side. Immunohistochemical localization of BDNF within the control-side olfactory bulb revealed small cells located mainly in the mitral cell layer and internal plexiform layer. Very few of the BDNF immunoreactive cells were visible in the bulb ipsilateral to the occlusion by P30. Given the roles of BDNF in survival of cells and plasticity during development, the decrease in BDNF expression subsequent to olfactory sensory deprivation may contribute to cellular and synaptic deficits observed by others following olfactory sensory deprivation.

Serotonin plays a permissive role in conditioned olfactory learning induced by norepinephrine in the neonate rat.

This study examined the role of serotonin (5-hydroxytryptamine; 5-HT) in conditioned olfactory learning in the rat pup. By injecting various combinations of the 5-HT(2A/2C) receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the beta adrenoceptor agonist, isoproterenol, we observed that isoproterenol alone acted as an unconditioned stimulus (US) to induce learning, as reported previously, whereas DOI alone did not induce learning. DOI combined with isoproterenol produced a leftward shift in the effective US dose such that suboptimal isoproterenol was effective, and previously optimal levels of isoproterenol were ineffective. Thus, 5-HT plays a facilitative but not an obligatory role in conditioned olfactory learning in the neonate rat.

Odor preference learning and memory modify GluA1 phosphorylation and GluA1 distribution in the neonate rat olfactory bulb: Testing the AMPA receptor hypothesis in an appetitive learning model

An increase in synaptic AMPA receptors is hypothesized to mediate learning and memory. AMPA receptor increases have been reported in aversive learning models, although it is not clear if they are seen with memory maintenance. Here we examine AMPA receptor changes in a cAMP/PKA/CREB-dependent appetitive learning model: odor preference learning in the neonate rat. Rat pups were given a single pairing of peppermint and 2 mg/kg isoproterenol, which produces a 24-h, but not a 48-h, peppermint preference in the 7-d-old rat pup. GluA1 PKA-dependent phosphorylation peaked 10 min after the 10-min training trial and returned to baseline within 90 min. At 24 h, GluA1 subunits did not change overall but were significantly increased in synaptoneurosomes, consistent with increased membrane insertion. Immunohistochemistry revealed a significant increase in GluA1 subunits in olfactory bulb glomeruli, the targets of olfactory nerve axons. Glomerular increases were seen at 3 and 24 h after odor exposure in trained pups, but not in control pups. GluA1 increases were not seen as early as 10 min after training and were no longer observed 48 h after training when odor preference is no longer expressed behaviorally. Thus, the pattern of increased GluA1 membrane expression closely follows the memory timeline. Further, blocking GluA1 insertion using an interference peptide derived from the carboxyl tail of the GluA1 subunit inhibited 24 h odor preference memory providing causative support for our hypothesis. PKA-mediated GluA1 phosphorylation and later GluA1 insertion could, conjointly, provide increased AMPA function to support both short-term and long-term appetitive memory.

Potentiation and prolongation of long-term odor memory in neonate rats using a phosphodiesterase inhibitor

Cyclic AMP has been shown to have a critical role in learning and memory in invertebrates. Here we use the rat pup odor preference learning model in which odor acts as a conditioned stimulus and beta-adrenoceptor stimulation acts as an unconditioned stimulus to test the role of cyclic AMP in an associative mammalian paradigm. A phosphodiesterase inhibitor that prevents cyclic AMP breakdown (cilomilast) makes a low, learning-ineffective dose of a beta-adrenoceptor agonist (isoproterenol, 1mg/kg) an effective unconditioned stimulus in pup odor preference learning. A dose of the phosphodiesterase inhibitor (cilomilast, 1 mg/kg) that induces learning with a weak unconditioned stimulus interferes with learning using a normally optimal unconditioned stimulus (isoproterenol, 2 mg/kg). Cilomilast (3 mg/kg) paired with peppermint odor during learning, prolonged memory at least four times longer than without the drug (24 h vs. 96 h). These data demonstrate a causal role for cyclic AMP in the acquisition and duration of odor preference learning in the rat pup.

Infusion of the metabotropic receptor agonist, DCG-IV, into the main olfactory bulb induces olfactory preference learning in rat pups

DCG-IV, a type 2 metabotropic glutamate receptor (mGluR2) agonist, was infused into the main olfactory bulb of 1-week-old pups exposed to peppermint odor. A preference for peppermint was demonstrated 24 h later. The data support the proposal that disinhibition at dendrodendritic synapses between granule cells and mitral cells is a critical component of olfactory memory formation in the rat pup olfactory bulb as well as in the accessory olfactory bulb of adult rodents.

PKA Increases in the Olfactory Bulb Act as Unconditioned Stimuli and Provide Evidence for Parallel Memory Systems: Pairing Odor with Increased PKA Creates Intermediate- and Long-Term, but Not Short-Term, Memories.

Neonatal odor-preference memory in rat pups is a well-defined associative mammalian memory model dependent on cAMP. Previous work from this laboratory demonstrates three phases of neonatal odor-preference memory: short-term (translation-independent), intermediate-term (translation-dependent), and long-term (transcription- and translation-dependent). Here, we use neonatal odor-preference learning to explore the role of olfactory bulb PKA in these three phases of mammalian memory. PKA activity increased normally in learning animals 10 min after a single training trial. Inhibition of PKA by Rp-cAMPs blocked intermediate-term and long-term memory, with no effect on short-term memory. PKA inhibition also prevented learning-associated CREB phosphorylation, a transcription factor implicated in long-term memory. When long-term memory was rescued through increased β-adrenoceptor activation, CREB phosphorylation was restored. Intermediate-term and long-term, but not short-term odor-preference memories were generated by pairing odor with direct PKA activation using intrabulbar Sp-cAMPs, which bypasses β-adrenoceptor activation. Higher levels of Sp-cAMPs enhanced memory by extending normal 24-h retention to 48-72 h. These results suggest that increased bulbar PKA is necessary and sufficient for the induction of intermediate-term and long-term odor-preference memory, and suggest that PKA activation levels also modulate memory duration. However, short-term memory appears to use molecular mechanisms other than the PKA/CREB pathway. These mechanisms, which are also recruited by β-adrenoceptor activation, must operate in parallel with PKA activation.

Mammalian intermediate-term memory: New findings in neonate rat

The ability of anisomycin, a translation inhibitor, and actinomycin, a transcription inhibitor to disrupt a cAMP/PKA-dependent odor preference memory in neonate rat was examined. Previous reports in invertebrates had described a novel translation-dependent intermediate-term memory dissected with these inhibitors, but similar effects have not been reported in mammalian memory systems. When anisomycin was infused into the olfactory bulb after the pairing of peppermint odor and the β-adrenoceptor agonist isoproterenol (2mg/kg), short-term memory (1 or 3h) was intact, but intermediate (5h) and long-term (24h) memory was disrupted. When actinomycin was infused, only long-term memory was disrupted. This pattern of results is consistent with that reported in invertebrates for intermediate-term memory and led us to try a lower level of the unconditioned stimulus (isoproterenol) to isolate intermediate-term memory from long-term memory. Pups given a dose of 1.5mg/kg isoproterenol paired with peppermint odor showed memory for peppermint 5h, but not 24h, after training. These observations in the rat pup olfactory system parallel short-, intermediate- and long-term memory characteristics previously described in invertebrates. Odor preference memory in neonate rodents offers a tool to increase our understanding of the properties and mechanisms of multi-phasic memory in mammals.

A phosphodiesterase inhibitor, cilomilast, enhances cAMP activity to restore conditioned odor preference memory after serotonergic depletion in the neonate rat.

In various learning and memory models, preventing the breakdown of cyclic adenosine monophosphate (cAMP) by using a phosphodiesterase (PDE) inhibitor promotes memory. In the rat pup odor preference learning model serotonin, acting through 5-HT(2A/C) receptors, has been shown to influence cAMP levels in the olfactory bulb initiated by beta-adrenoceptor activation, as also seen in the neocortex. Since depletion of olfactory bulb serotonin prevents learning in the rat pup odor preference model, we ask whether a PDE inhibitor could restore that learning and also examined the influence of these manipulations on the temporal bulbar cAMP signal associated with successful learning. In this study, we found that a PDE4 inhibitor overcame learning deficits seen 24h after a 10min training trial on postnatal day 6 using the beta-adrenoceptor agonist, isoproterenol as the unconditioned stimulus. We found in a previous study, that use of a PDE4 inhibitor during learning in normal pups extended memory to more than 48h. However, in the present study the PDE4 treatment did not enable this memory extension in 5-HT depleted pups. An increase in the cAMP signal at the end of the 10min training trial occurred in the presence of the PDE4 inhibitor. Such a cAMP increase has been associated with successful learning and is normally absent with bulbar 5-HT depletion. These results suggest PDE4 inhibitors may be useful therapeutically in disorders associated with reductions in serotonergic function.

Lack of evidence for serotonergic effect on the cellular development of the olfactory bulb in the postnatal rat.

Serotonin has been postulated to influence several developmental parameters. The potential role of serotonin in the development of the rat olfactory bulb, a simple cortical structure, was determined following selective depletion of serotonin to the olfactory bulb of neonate rats. In the neonate, 5,7-dHT was injected into the anterior olfactory nucleus to selectively destroy serotonergic axons leading to the bulb. Following survival of 5 days to 3 months, the rats were sacrificed and analyzed by immunocytochemical markers, Nissl stain, Golgi impregnation, and image analysis. The serotonin depletions had no significant effect on the cytoarchitecture of the bulb or on neuronal or glial cell growth. In addition, the depletions did not affect neuronal migration or differentiation (overall length of dendrites, branch points, or dendritic spines) of cell populations in the bulb. These findings suggest that serotonin does not, by itself, affect the overall development of cellular elements in the bulb, although this study does not rule out the possibility that serotonin may affect other parameters of development.