John H. McLean

Increased β adrenoceptor activation overcomes conditioned olfactory learning deficits induced by serotonin depletion

It was hypothesized that 5-HT2 receptors in the olfactory bulb prime the bulbar response to a beta adrenoceptor mediated unconditioned stimulus (UCS) during odor preference learning in 1-week-old rat pups. The ability of 4 mg/kg of isoproterenol + stroking and 6 mg/kg of isoproterenol + no stroking to induce normal odor preference learning in pups depleted of bulbar 5-HT in the present study supports the hypothesis. The inverted-U curve relation between UCS strength and learning also appears to occur within the bulb.

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.

Selective lesions of neural pathways following viral inoculation of the olfactory bulb.

In the present study, herpes simplex virus type 1 (HSV1) was injected into the olfactory bulb of the rat in order to determine the impact of viral infection on neural pathways, neurotransmitters, and behavior. In many animals, these injections caused considerable neuronal loss in regions that project to the bulb including the primary olfactory cortex and locus coeruleus (LC). Short-term (2-5 days postinjection) studies using immunocytochemical colocalization of virus and transmitter markers showed that cholinergic (ACh) neurons in the horizontal nucleus of the diagonal band, serotonergic (5-HT) neurons in the dorsal and median raphe nuclei, and noradrenergic (NE) neurons in the LC became infected with virus. Almost all NE neurons in the ipsilateral LC were infected while a smaller proportion of 5-HT and ACh neurons in their respective nuclei contained virus. In order to determine long-term effects of viral infection, virus injection into the olfactory bulb was followed by antiviral treatment and sacrifice 17 days to 7 months postinjection. Quantitative analysis of selected cortical regions (olfactory bulb, cingulate cortex, parietal cortex) revealed decreased NE-immunoreactive fibers while 5-HT axons from the dorsal and median raphe nuclei were not significantly affected. No changes in acetylcholinesterase staining in these cortical regions were observed, indicating that cholinergic axons were not significantly changed. Ten of the 36 animals that survived long-term after HSV1 inoculation were also tested in a water maze task before sacrifice to determine if the viral infection was associated with spatial learning deficits. Spatial learning deficits correlated with the degree of primary olfactory cortex damage but not with 5-HT, NE, or ACh axon losses.

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.

Anterograde transport of HSV-1 and HSV-2 in the visual system

The anterograde spread of herpesvirus in the visual system subsequent to retinitis has been observed clinically. We compared the ability of two well-studied Herpes simplex virus (HSV) strains to be transported in the anterograde direction in the hamster visual system: strain McIntyre, representing HSV-1, and strain 186, representing HSV-2. Intravitreal injection of HSV-2 labeled more retinorecipient neurons than did HSV-1, suggesting important type differences in the ability of HSV to infect retinorecipient neurons after intravitreal injection. The most likely explanation for our results is that HSV-2 is more efficiently adsorbed than HSV-1 in the retinal ganglion cells. Our results also suggest that HSV may be useful as an anterograde transneuronal tracer for neuroanatomical studies of the visual system.

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.