Calcineurin inhibition eliminates the normal inverted U curve, enhances acquisition and prolongs memory in a mammalian 3'-5'-cyclic AMP-dependent learning paradigm.

The role protein phosphatase 2B (calcineurin, CaN) plays in learning and memory has received a significant amount of attention due to its promotion of the dephosphorylation of 3'-5'-cyclic AMP response element binding protein (CREB). Researchers have ascertained that overexpression of CaN is associated with memory retention deficits [Foster TC, Sharrow KM, Masse JR, Norris CM, Kumar A (2001) Calcineurin links Ca(2+) dysregulation with brain aging. J Neurosci 21:4066-4073; Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39-49], while CaN inhibition enhances learning and memory [Gerdjikov TV, Beninger RJ (2005) Differential effects of calcineurin inhibition and protein kinase A activation on nucleus accumbens amphetamine-produced conditioned place preference in rats. Eur J Neurosci 22:697-705; Ikegami S, Inokuchi K (2000) Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction. Neuroscience 98:637-646]. The present study hypothesized that infusion of a CaN inhibitor (FK506) bilaterally into the olfactory bulbs of postnatal day 6 Sprague Dawley rat pups would prolong the duration of a conditioned odor preference and retard cyclic AMP response element binding protein dephosphorylation. A 2 mg/kg s.c. injection of isoproterenol (ISO, beta-adrenoceptor agonist) was paired with a 10 min exposure to peppermint and subsequently an infusion of FK506. Immunohistochemistry for phosphorylated 3'-5'-cyclic AMP response element binding protein (pCREB) revealed that unilateral infusion of FK506 resulted in an amplification of phosphorylated CREB in the olfactory bulb 40 min after training compared with saline-infused bulbs. Pups infused bilaterally with FK506 maintained a learned preference for peppermint 48, 72 and 96 h after training. CaN inhibition also modified the conventional inverted U curve obtained when ISO is used to replace stroking, as the unconditioned stimulus. When pups were infused with FK506, learning occurred with sub- and supra-optimal doses of ISO indicating that CaN overcomes non-optimal effects ISO may have on learning. We demonstrate that CaN inhibition can extend the duration of conditioned olfactory memory and may provide a target for memory prolongation that is superior to even phosphodiesterase inhibition observed in previous studies.

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.

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.

Isoproterenol increases CREB phosphorylation and olfactory nerve-evoked potentials in normal and 5-HT-depleted olfactory bulbs in rat pups only at doses that produce odor preference learning.

Norepinephrine (NE) and serotonin (5-HT) are important modulators of early odor preference learning. NE can act as an unconditioned stimulus (UCS), whereas 5-HT facilitates noradrenergic actions. In this study, we examined the phosphorylation of an important transcription factor, cAMP response element binding protein (CREB), which has been implicated in long-term-memory formation (McLean et al. 1999) during NE-induced odor preference learning in normal and olfactory bulb 5-HT-depleted rat pups. We also examined NE modulation of olfactory nerve-evoked field potentials (ON-EFPs) in anesthetized normal and bulbar 5-HT depleted pups. Systemic injection of 2 mg/kg isoproterenol (beta-adrenoceptor agonist) induced odor preference learning, enhanced pCREB expression in the olfactory bulbs at 10 min after odor pairing, and increased ON-EFPs in normal rat pups but not in bulbar 5-HT-depleted rat pups. A dose of 6 mg/kg isoproterenol, which was ineffective in modulating these measures in normal rat pups, induced odor preference learning, enhanced phosphorylated CREB (pCREB) expression, and increased ON-EFPs in bulbar 5-HT-depleted pups. These outcomes suggest that NE and 5-HT promote specific biochemical and electrophysiological changes that may critically underlie odor preference learning.

pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training.

Early olfactory preference learning in rat pups occurs when novel odors are paired with tactile stimulation, for example stroking. cAMP-triggered phosphorylation of cAMP response element binding protein (pCREB) has been implicated as a mediator of learning and memory changes in various animals (Frank and Greenberg 1994). In the present study we investigate whether CREB is phosphorylated in response to conditioned olfactory training as might be predicted given the proposed role of the phosphorylated protein in learning. On postnatal day 6, pups were trained for 10 min using a standard conditioned olfactory learning paradigm in which a conditioned stimulus, Odor, was either used alone or paired with an unconditioned stimulus, Stroking (using a fine brush to stroke the pup). In some instances stroking only was used. The pups were sacrificed at 0, 10, 30, or 60 min after the training. Using Western blot analysis, we observed that the majority of olfactory bulbs in conditioned pups (Odor + Stroking) had a greater increase in pCREB activation at 10 min after training than pups given nonlearning training (Odor only or Stroking only). The phosphorylated protein levels were low at 0 min and at 60 min after training. This is in keeping with the slightly delayed and short-lived activation period for this protein. The localization of pCREB increases within the olfactory bulb as seen by immunocytochemistry. Naive pups were not exposed to odor or training. There was a significantly higher level of label in mitral cell nuclei within the dorsolateral quadrant of the bulb of pups undergoing odor-stroke pairing. No significant differences were observed among nonlearning groups (Naive, Odor only, or Stroking only) or among any training groups in the granule or periglomerular cells of the dorsolateral region. The localized changes in the nuclear protein are consistent with studies showing localized changes in the bulb in response to a learned familiar odor. The present study demonstrates that selective increases in pCREB occur as an early step following pairing procedures that normally lead to the development of long-term olfactory memories in rat pups. These results support the hypothesized link between pCREB and memory formation.

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.

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.

Loss of cortical serotonin2A signal transduction in senescent rats: reversal following inhibition of protein kinase C.

Using grease gap recordings, age-related changes in serotonin2A receptors were assessed in sensorimotor regions of the cortex by examining serotonin-induced facilitation of the N-methyl-D-aspartate depolarization in cortical wedges prepared from young adult (3-6 months) and senescent (22-34 months) Fisher 344 rats. Serotonin (10-100 microM) facilitated the N-methyl-D-aspartate depolarization in wedges from young adult rats in a concentration-dependent manner, whereas no facilitation was observed in wedges from senescent rats. Similar results were obtained when +/- 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, a mixed serotonin2A and serotonin2C receptor agonist, was substituted for serotonin. In contrast, agonists at alpha 1A-adrenoceptors, metabotropic glutamate receptors and muscarinic cholinoceptors facilitated the N-methyl-D-aspartate depolarization in wedges from both young adult and senescent rats. Chelerythrine and staurosporine, inhibitors of protein kinase C, but not concanavalin A, myo-inositol or calmodulin antagonists, restored the serotonin facilitation in wedges from senescent animals. In situ hybridization histochemistry revealed that serotonin2A receptor messenger RNA was present in layers II-VI of the cortex, with the highest density of silver grains located in layers III and V of both young adult and senescent rats. Detailed examination of layer V showed that silver grains were significantly higher than background only over pyramidal cells. We conclude that serotonin2A receptors are expressed by pyramidal cells in both young adult and senescent rats and that serotonin acts directly on these receptors to facilitate the N-methyl-D-aspartate depolarization. Moreover, in senescent rats, signal transduction at cortical serotonin2A receptors involved with facilitation of the N-methyl-D-aspartate response is compromised as a result of protein kinase C activation.

Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat.

The olfactory bulb receives a dense serotonergic input and appears to require serotonergic input in early olfactory associational learning. However, it is not known which cell types receive the serotonergic input or whether the cells express markers for the input throughout life. These issues need resolution in order for the mechanisms of serotonergic interactions to be better understood. The mRNA for the 5-HT2A receptor was localized in the olfactory bulb of postnatal day 1, 2, 14 and 9-month-old Sprague-Dawley rats as well as in the bulb of adult (6 months) and aged (22-30 month) Fisher 344 rats by in situ hybridization using an 35S-labelled 5-HT2A- specific oligonucleotide probe mixture. In all animals, hybridization was observed in mitral cells which are the major output cells of the main olfactory bulb. Tufted cells, located in the external plexiform layer and juxtaglomerular region, were readily observed in adult and aged animals and were also observed, albeit not as readily, in neonate pups. Quantitative analysis of the silver grain density over cells confirmed qualitative observations and showed that mitral and tufted cells were labelled in the neonate as well as in adult and aged animals. Labelled cells were also numerous in the external division of the anterior olfactory nucleus in all animals. 5-HT2A receptor mRNA could not be detected either qualitatively or quantitatively by in situ hybridization in the accessory olfactory bulb, nor could it be observed in the olfactory epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Serotonergic influence on olfactory learning in the neonate rat.

The role of the serotonergic innervation of the olfactory bulb was examined in neonate rat pups (Sprague-Dawley) by utilizing an olfactory learning paradigm (Sullivan, McGaugh, & Leon, 1991; Sullivan, Wilson, & Leon, 1989). One hundred fifty nanoliters of the neurotoxin 5,7-dihydroxytryptamine (5,7-dHT, 10 micrograms/microliters) was injected into each anterior olfactory nucleus of Postnatal Day 2 (PND 2) pups in order to selectively deplete the serotonergic (5-HT) innervation of the olfactory bulb. On PND 8, control, sham-operated, or 5-HT-depleted pups were presented with stroke (unconditioned stimulus) paired with peppermint odor (conditioned stimulus) or were presented with peppermint alone. Other pups remained naive, i.e., they were not presented with either stroke or odor. The following day, the pups were placed on a mesh screen above two boxes containing either fresh bedding or bedding with peppermint odor for five 1-min trials. Control and sham-operated pups that were previously trained by the odor/stroke paradigm spent significantly more time over the peppermint odor than similarly trained 5-HT-depleted pups, pups trained using the odor only paradigm, or naive pups. Immunocytochemistry verified that the 5,7-dHT injections specifically depleted the 5-HT innervation of the olfactory bulb and left the 5-HT innervation of more caudal levels of the neuraxis (e.g., piriform cortex) intact. The locomotor behavior of the pups was not affected by the 5-HT depletions. This study is the initial investigation to suggest that 5-HT innervation to the olfactory bulb is important in the acquisition or expression of olfactory-based learned behavior in the neonate rat.