Edwin J. Barea-Rodriguez

Role of Hippocampal CA3 μ-Opioid Receptors in Spatial Learning and Memory

The dorsal CA3 region of the hippocampus is unique in its connectivity, sensitivity to neurotoxic lesions, and its ability to encode and retrieve episodic memories. Computational models of the CA3 region predict that blocking mossy-fiber and/or perforant path activity to CA3 would cause impairments in learning and recall of spatial memory, respectively. Because the CA3 region contains μ-opioid receptors and receives inputs from the mossy-fiber and lateral perforant pathways, both of which contain and release opioid peptides, we tested the hypothesis that inactivating μ-opioid receptors in the CA3 region would cause spatial learning and memory impairments and retrieval deficits. In this study, male Sprague Dawley rats were trained in a Morris water maze after a single bilateral intrahippocampal injection of either saline or the selective and irreversible μ-opioid receptor antagonist β-funaltrexamine (β-FNA) into area CA3. We found that μ-opioid receptor binding decreased 24 hr after β-FNA injection and returned to control levels 11 d after injection. Injections of β-FNA into the CA3 region, but not into the ventricles, caused a significant impairment in the acquisition of spatial learning without causing sensory or motor deficits. New learning was not affected once μ-opioid receptor levels replenished (>11 d after injection). In pretrained animals, β-FNA significantly impaired spatial memory retrieval and new (reversal) learning. These data are consistent with theoretical models of CA3 function and suggest that CA3 μ-opioid receptors play an important role in the acquisition and retrieval of spatial memory.

Water maze training in aged rats: effects on brain metabolic capacity and behavior

The effects of Morris water maze training on brain metabolism and behavior were compared between aged (20-22 months) and young (2-4 months) Fischer 344 male rats. Each group had yoked controls, which swam the same amount of time as the trained rats but without the platform. This was followed after 9 days by quantitative histochemical mapping of brain cytochrome oxidase, the terminal enzyme for cellular respiration. The aged rats spent a significantly lower percent of time in the correct quadrant and had a longer latency to escape to the hidden platform, relative to the young rats. Metabolic differences between trained aged and young rats were found in regions related to escape under stress: perirhinal cortex, basolateral amygdala and lateral habenula; and vestibular nuclei that guide orientation in three-dimensional space. These differences were not found in the yoked swimming rats. The results suggest that, at the time point investigated, water maze training in aged Fischer 344 rats produces altered oxidative energy metabolism in task-relevant limbic and vestibular regions.

Opioid receptor modulation of mossy fiber synaptogenesis: Independence from long-term potentiation

Long-term potentiation (LTP) at the mossy fiber-CA3 synapse of the rat hippocampus is an NMDA receptor-independent form of synaptic plasticity that is sensitive to opioid receptor antagonists [12]. In the present study, Timms stain, a zinc detecting histological marker commonly used to infer synaptogenesis in the mossy fiber projection, was used to examine whether synaptogenesis occurs in response to mossy fiber LTP induction in the adult rat in vivo. Seven days following the induction of mossy fiber LTP by non-seizure-inducing high-frequency stimulation of the mossy fibers, a prominent band of Timms staining appeared bilaterally in the infrapyramidal region of the stratum oriens in area CA3. Staining was more prominent on the side contralateral to the stimulation. Systemic administration of the opioid receptor antagonist naloxone, sufficient to block mossy fiber LTP induction, did not block the development of Timms staining in the infrapyramidal region ipsilateral to stimulation, but it did block stimulation-induced increases in Timms staining observed contralaterally. Systemic administration of (+/-) CPP, a competitive NMDA receptor-antagonist, by contrast, did not block the induction of LTP and did not alter the increase in Timms staining observed either ipsilaterally or contralaterally. The increase in Timms staining in the infrapyramidal region suggests that mossy fiber synaptogenesis occurs in response to non-seizure inducing stimulation. Synaptogenesis does not appear to be directly related to opioid receptor-dependent mossy fiber LTP induction, because it occurs in the presence of naloxone which blocks LTP. The mossy fiber synaptogenesis occurring contralaterally appears to be regulated by endogenous opioid peptides, because it is blocked by naloxone.

Learning associated increase in heat shock cognate 70 mRNA and protein expression.

The Morris water maze is a task widely used to investigate cellular and molecular changes associated with spatial learning and memory. This task has both spatial and aversive (swimming related stress) components. It is possible that stress may influence cellular modifications observed after learning the Morris water maze spatial task. Heat shock proteins, also known as stress proteins, are up-regulated in response to thermal stress, trauma, or environmental insults. In the rat hippocampus, psychophysiological stress increases the levels of heat shock protein 70 (HSC70). In this study, we investigated whether the expression of the hsc70 gene is modulated in the hippocampus during learning of the Morris water maze task. Five groups of rats were trained in the Morris water maze task for varying amounts of time (either 1, 2, 3, 4, or 5 days). Training consisted of 10 trials/day in which the animals were given 60s to find a submerged platform. Rats were sacrificed 24h after their last training trial. Results showed a significant increase in hsc70 mRNA and protein levels in the hippocampal formation after two and three days of training, respectively. The increase in mRNA and protein was associated with learning but not stress because the increase was not observed in the yoked control animals. These findings suggest that cellular and molecular changes can occur independent of stress. Moreover, the results are the first to implicate hsc70 expression in spatial learning.

ERK phosphorylation is required for retention of trace fear memory

The extracellular signal-regulated kinase (ERK) has been previously associated with long-term memory formation. Earlier studies have demonstrated a role for phospho-ERK in delay fear conditioning and it has been shown to disrupt trace fear memory when inhibited after training. cAMP response element binding protein (CREB) is a key transcription factor that has been implicated in long-term memory formation across different species. It has also been shown to be modulated by ERK. In our study, we used the drug SL327 to prevent ERK phosphorylation. Two groups of Fischer 344 male rats (2-4 months) were injected intraperitoneally with 100% DMSO (2 ml/kg) or SL327 (100 mg/kg/2 ml dissolved in DMSO) 45 min before 10 trials of trace fear conditioning. Each trial consisted of a tone paired with a footshock with a 30-s interval separating the stimuli. Twenty-four hours later, rats were tested for fear to the tone. Our results showed that SL327-treated rats displayed memory deficits 24 h after training. Western blot analyses of total hippocampal protein revealed a significant increase in phosphorylated ERK immediately after training. There were also decreases in phosphorylated ERK at 45 and 90 min post-injection of SL327-treated rats as compared to DMSO-treated rats, but levels of phosphorylated CREB remained the same. These findings indicate that ERK phosphorylation is increased immediately after trace fear conditioning and inhibiting this increase is correlated with memory deficits in trace fear conditioning 24 h later. These findings support a role for ERK phosphorylation in the formation of trace fear memories.

p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength.

During muscle development, the p75NTR is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75NTR‐deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75NTR was detected on myoblasts but the high affinity NGF receptor, trk A, was not co‐expressed with p75NTR. In vitro, proliferating C2C12 and primary myoblasts co‐expressed the p75NTR and MyoD, but immunofluorescent analysis of primary myoblasts and RT‐PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75NTR in neurons, p75NTR‐positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I‐κB and AKT, the cells contained increased levels of both phospho‐proteins, both of which promote cell survival. By contrast, neurotrophin‐treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75NTR mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.

Brief Novelty Exposure Facilitates Dentate Gyrus LTP in Aged Rats

Aging is associated with a decreased capacity for dentate gyrus (DG) granule cell depolarization as well as reduced perforant path activation. Although it is well established that the maintenance of DG long‐term potentiation (LTP) over days is impaired in aged, as compared to young animals, the threshold for inducing this LTP has never been investigated in aged, awake animals. In addition, although exposure to novelty prior to θ‐burst stimulation (TBS) increases both the induction and longevity of DG LTP in adult rats, the effects of exposure to novelty on LTP in aged rats have never been investigated. Here, we report that although TBS delivered in the home cage induces robust and long‐lasting DG LTP in young rats, TBS fails to induce DG LTP in aged rats. Interestingly, delivery of TBS to aged rats exploring novel environments induces robust and long‐lasting LTP, with the induction, but not the longevity, of this LTP being similar in magnitude to that observed in young rats delivered TBS in the home cage. These results indicate that although TBS‐induced DG LTP is impaired in aged, as compared to young rats, TBS during exploration of novel environments is sufficient to rescue age‐related deficits in DG LTP. We discuss these observations in the context of previous findings suggesting that the facilitation of LTP by exposure to novel environments results as a consequence of reduced network inhibition in the DG and we suggest that, in spite of age‐related changes in the DG, this capacity persists in aged rats and represents a nondietary and nonpharmacological way to facilitate DG LTP during aging.

Fischer 344 rats display age-related memory deficits in trace fear conditioning.

A functional hippocampus is required for trace fear conditioning, which involves learning the association of a tone and shock that are separated over time. Young and aged rats received 10 trace conditioning trials. Twenty-four hours later, rats were tested for fear to the tone in a novel chamber by measuring freezing. The results showed significantly lower levels of freezing in aged rats as compared with young rats, which provides evidence of age-related memory impairments. Pseudorandom conditioning groups showed low levels of freezing, indicative of no associative memory. Age-related memory deficits were not found with delay conditioning, which suggests no age-related sensory-motor deficits. These data suggest that aging hinders the ability of the hippocampus to process information separated over time.

The CC chemokine receptor 5 regulates olfactory and social recognition in mice.

Chemokines are chemotactic cytokines that regulate cell migration and are thought to play an important role in a broad range of inflammatory diseases. The availability of chemokine receptor blockers makes them an important therapeutic target. In vitro, chemokines are shown to modulate neurotransmission. However, it is not very clear if chemokines play a role in behavior and cognition. Here we evaluated the role of CC chemokine receptor 5 (CCR5) in various behavioral tasks in mice using Wt (Ccr5⁺/⁺) and Ccr5-null (Ccr5⁻/⁻)mice. Ccr5⁻/⁻ mice showed enhanced social recognition. Administration of CC chemokine ligand 3 (CCL3), one of the CCR5-ligands, impaired social recognition. Since the social recognition task is dependent on the sense of olfaction, we tested olfactory recognition for social and non-social scents in these mice. Ccr5⁻/⁻ mice had enhanced olfactory recognition for both these scents indicating that enhanced performance in social recognition task could be due to enhanced olfactory recognition in these mice. Spatial memory and aversive memory were comparable in Wt and Ccr5⁻/⁻ mice. Collectively, these results suggest that chemokines/chemokine receptors might play an important role in olfactory recognition tasks in mice and to our knowledge represents the first direct demonstration of an in vivo role of CCR5 in modulating social behavior in mice. These studies are important as CCR5 blockers are undergoing clinical trials and can potentially modulate behavior.

Long-Term Potentiation, Long-Term Depression, and Learning

Publisher Summary Almost everyone agrees that information is acquired, stored, and retrieved by the brain. All brains consist of individual cellular elements. Most neurons have the same parts: a dendritic tree, cell body, axon, and synaptic boutons. From the time of Pavlov, conditioned reflexes have been thought to involve specific neural pathways. In fact, simple neural reflexes may be incorporated into conditioned reflexes. The hippocampus is viewed as having a temporally restricted role in memory, in both animals and humans. As mentioned in this chapter with respect to the saturation experiments of McNaughton and colleagues, when viewed from the perspective of distributed memories, partial sparing of function may be sufficient to permit learning. One of the reasons to target genes is that these genetic procedures have the potential to overcome the current limitations of pharmacology. The hippocampus displays obvious anatomical abnormalities, including an increase in granule and pyramidal cells. The results of the random probe test suggest that the mutant mice did not know the spatial location of the hidden platform, although they apparently were able to use some strategy to escape the maze.