Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA.

The astrocyte brain fatty acid binding protein (Fabp7) has previously been shown to have a coordinated diurnal regulation of mRNA and protein throughout mouse brain, and an age-dependent decline in protein expression within synaptoneurosomal fractions. Mechanisms that control time-of-day changes in expression and trafficking Fabp7 to the perisynaptic process are not known. In this study, we confirmed an enrichment of Fabp7 mRNA and protein in the astrocytic perisynaptic compartment, and observed a diurnal change in the intracellular distribution of Fabp7 mRNA in molecular layers of hippocampus. Northern blotting revealed a coordinated time-of-day-dependent oscillation for the Fabp7 mRNA poly(A) tail throughout murine brain. Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) regulates subcellular trafficking and translation of synaptic plasticity-related mRNAs. Here we show that Fabp7 mRNA coimmunoprecipitated with CPEB1 from primary mouse astrocyte extracts, and its 3'UTR contains phylogenetically conserved cytoplasmic polyadenylation elements (CPEs) capable of regulating translation of reporter mRNAs during Xenopus oocyte maturation. Given that Fabp7 expression is confined to astrocytes and neural progenitors in adult mouse brain, the synchronized cycling pattern of Fabp7 mRNA is a novel discovery among known CPE-regulated transcripts. These results implicate circadian, sleep, and/or metabolic control of CPEB-mediated subcellular trafficking and localized translation of Fabp7 mRNA in the tripartite synapse of mammalian brain.

Cytoplasmic to nuclear localization of fatty-acid binding protein correlates with specific forms of long-term memory in Drosophila.

We recently reported evidence implicating fatty-acid binding protein (Fabp) in the control of sleep and memory formation. We used Drosophila melanogaster to examine the relationship between sleep and memory through transgenic overexpression of mouse brain-Fabp, Fabp7, or the Drosophila Fabp homolog, (dFabp). The key findings are that 1) a genetically induced increase in daytime consolidated sleep (naps) correlates with an increase in cognitive performance, and 2) a late "window" of memory consolidation occurs days after the traditionally understood "synaptic" consolidation. Exactly how Fabp-signaling may be involved in converting normal to enhanced long-term memory (LTM) is not known. Here we describe additional data which support relative subcellular compartmental localization of Fabp in regulating stage associations of different forms of memory in Drosophila. Anesthesia resistant memory (ARM) is a longer lasting memory that is produced by massed training, but unlike LTM produced by spaced training, it is insensitive to protein synthesis inhibitors and does not persist as long. We observed that the ratio of ARM to LTM performance index of Fabp7-transgenic flies is proportional to the relative cytoplasmic to nuclear Fabp7 expression level. These data suggest a common lipid-signaling cascade exists between phases of memory formation previously thought to be molecularly distinct.

Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila.

Sleep is thought to be important for memory consolidation, since sleep deprivation has been shown to interfere with memory processing. However, the effects of augmenting sleep on memory formation are not well known, and testing the role of sleep in memory enhancement has been limited to pharmacological and behavioral approaches. Here we test the effect of overexpressing the brain-type fatty acid binding protein (Fabp7) on sleep and long-term memory (LTM) formation in Drosophila melanogaster. Transgenic flies carrying the murine Fabp7 or the Drosophila homologue dFabp had reduced baseline sleep but normal LTM, while Fabp induction produced increases in both net sleep and LTM. We also define a post-training consolidation "window" that is sufficient for the observed Fabp-mediated memory enhancement. Since Fabp overexpression increases consolidated daytime sleep bouts, these data support a role for longer naps in improving memory and provide a novel role for lipid-binding proteins in regulating memory consolidation concurrently with changes in behavioral state.

The dendritically targeted protein Dendrin is induced by acute nicotine in cortical regions of adolescent rat brain.

The response of the brain to addictive substances such as nicotine includes the rapid induction of genes that influence synaptic events. This response is different in adolescent brain, which continues to undergo synaptic remodeling in regions that include reward-associated corticolimbic areas. We report here that acute nicotine (0.4 mg/kg), but not cocaine or exposure to a novel environment, induces the expression of the dendritically targeted, corticolimbic mRNA Dendrin in specific regions of adolescent brain. Acute nicotine resulted in an increase in Dendrin mRNA levels in the adolescent prefrontal cortex that was not evident in adult animals. The induction in Dendrin mRNA was a rapid, short-lived transcriptional event that resulted in changes in Dendrin protein. For example, an increase in Dendrin protein levels following nicotine treatment paralleled enhanced Dendrin immunoreactivity in the dendrites of pyramidal neurons of somatosensory cortex. As Dendrin is an important component of cytoskeletal modifications at the synapse, these results suggest that nicotine influences unique plasticity-related changes in the adolescent forebrain that differ from the adult.

Brain fatty acid binding protein (Fabp7) is diurnally regulated in astrocytes and hippocampal granule cell precursors in adult rodent brain.

Brain fatty acid binding protein (Fabp7), which is important in early nervous system development, is expressed in astrocytes and neuronal cell precursors in mature brain. We report here that levels of Fabp7 mRNA in adult murine brain change over a 24 hour period. Unlike Fabp5, a fatty acid binding protein that is expressed widely in various cell types within brain, RNA analysis revealed that Fabp7 mRNA levels were elevated during the light period and lower during dark in brain regions involved in sleep and activity mechanisms. This pattern of Fabp7 mRNA expression was confirmed using in situ hybridization and found to occur throughout the entire brain. Changes in the intracellular distribution of Fabp7 mRNA were also evident over a 24 hour period. Diurnal changes in Fabp7, however, were not found in postnatal day 6 brain, when astrocytes are not yet mature. In contrast, granule cell precursors of the subgranular zone of adult hippocampus did undergo diurnal changes in Fabp7 expression. These changes paralleled oscillations in Fabp7 mRNA throughout the brain suggesting that cell-coordinated signals likely control brain-wide Fabp7 mRNA expression. Immunoblots revealed that Fabp7 protein levels also underwent diurnal changes in abundance, with peak levels occurring in the dark period. Of clock or clock-regulated genes, the synchronized, global cycling pattern of Fabp7 expression is unique and implicates glial cells in the response or modulation of activity and/or circadian rhythms.

Food-associated cues alter forebrain functional connectivity as assessed with immediate early gene and proenkephalin expression.

BACKGROUND: Cues predictive of food availability are powerful modulators of appetite as well as food-seeking and ingestive behaviors. The neurobiological underpinnings of these conditioned responses are not well understood. Monitoring regional immediate early gene expression is a method used to assess alterations in neuronal metabolism resulting from upstream intracellular and extracellular signaling. Furthermore, assessing the expression of multiple immediate early genes offers a window onto the possible sequelae of exposure to food cues, since the function of each gene differs. We used immediate early gene and proenkephalin expression as a means of assessing food cue-elicited regional activation and alterations in functional connectivity within the forebrain. RESULTS: Contextual cues associated with palatable food elicited conditioned motor activation and corticosterone release in rats. This motivational state was associated with increased transcription of the activity-regulated genes homer1a, arc, zif268, ngfi-b and c-fos in corticolimbic, thalamic and hypothalamic areas and of proenkephalin within striatal regions. Furthermore, the functional connectivity elicited by food cues, as assessed by an inter-regional multigene-expression correlation method, differed substantially from that elicited by neutral cues. Specifically, food cues increased cortical engagement of the striatum, and within the nucleus accumbens, shifted correlations away from the shell towards the core. Exposure to the food-associated context also induced correlated gene expression between corticostriatal networks and the basolateral amygdala, an area critical for learning and responding to the incentive value of sensory stimuli. This increased corticostriatal-amygdalar functional connectivity was absent in the control group exposed to innocuous cues. CONCLUSION: The results implicate correlated activity between the cortex and the striatum, especially the nucleus accumbens core and the basolateral amygdala, in the generation of a conditioned motivated state that may promote excessive food intake. The upregulation of a number of genes in unique patterns within corticostriatal, thalamic, and hypothalamic networks suggests that food cues are capable of powerfully altering neuronal processing in areas mediating the integration of emotion, cognition, arousal, and the regulation of energy balance. As many of these genes play a role in plasticity, their upregulation within these circuits may also indicate the neuroanatomic and transcriptional correlates of extinction learning.

The zinc-binding protein chordc1 undergoes complex diurnal changes in mRNA expression during mouse brain development.

Diurnal changes in Chordc1 mRNA were recently described in mouse hypothalamus. This report shows that Chordc1 mRNA changes rhythmically throughout the entire adult brain with highest expression levels occurring around the dark-light transition. The rhythmic cycling pattern of Chordc1 was retained under various light-dark schedules and analysis of adult whole brain revealed diurnal patterns that were different than young animals (postnatal day (P) 6). Analysis of adult hippocampus, prefrontal cortex and cerebellum confirmed these observations and a comparison between adult and P6 animals using in situ hybridization indicated that Chordc1 underwent coordinated but altered diurnal changes in mRNA abundance during development. Further, a developmental profile of Chordc1 expression beginning at embryonic day 17 revealed a regional distribution of Chordc1 consistent with its adult pattern. These results suggest that Chordc1 mRNA is under complex and widespread transcriptional regulation during development and implicate Chordc1 in circadian and/or homeostatic mechanisms in mammalian brain.

Acute stress and nicotine cues interact to unveil locomotor arousal and activity-dependent gene expression in the prefrontal cortex.

BACKGROUND: This study examines the interactive effects of acute stress and nicotine-associated contextual cues on locomotor activity and activity-dependent gene expression in subregions of the prefrontal cortex. METHODS: Locomotor activity of rats was measured in a context associated with either low-dose nicotine or saline administration with or without 5 minutes of pre-exposure to ferrets, a nonphysical stressor. After 45 minutes in the test environment, plasma corticosterone levels and mRNA levels of the immediate-early genes Arc, NGFI-B, and c-Fos in prefrontal and primary motor cortical subregions were measured. RESULTS: Stress alone increased plasma corticosterone and prefrontal cortex gene expression. Low-dose nicotine cues had no effect on corticosterone levels nor did they elicit conditioned motor activation, and they caused minor elevations in gene expression. Stress and low-dose nicotine cues, however, interacted to elicit conditioned motor activation and further increases in early response gene expression in prefrontal but not in the primary motor cortical subregions. CONCLUSIONS: Stress interacts with nicotine-associated cues to uncover locomotor arousal, a state associated with prefrontal neuronal activation and immediate early gene expression. Thus, in nicotine-experienced individuals, stress may be an important determinant of subjective reactivity and prefrontal cortex activation that occurs in response to nicotine-associated cues.

Expression of the transcriptional coactivator CITED1 in the adult and developing murine brain.

The transcription coactivator CITED1 is an important mediator of transcriptional events regulated by estrogen or TGF-beta. We used in situ hybridization to delineate the distribution of CITED1 mRNA in the adult and developing murine brain and found robust CITED1 expression in ventral hypothalamus and midbrain raphe. The distribution of CITED1 in these regions overlapped the reported expression of estrogen receptors alpha and beta. Less intense expression of CITED1 was also evident in medial preoptic area, subfornical organ, thalamus and cerebral cortex. CITED1 mRNA in the arcuate nucleus (an area of active transcriptional modulation by TGF-beta) was evident in postmigratory neurons as early as embryonic day 16. Expression of CITED1 in arcuate continued throughout postnatal development. CITED1 in developing cerebellum was first evident in external granule cells and was transiently expressed in the Purkinje cell/granule cell layer in a temporal pattern similar to estrogen receptor-beta. The spatial and temporal distribution of CITED1 mRNA reported here is consistent with a role for CITED1 in the modulation of transcriptional events mediated by steroid hormone and cytokine signaling pathways.

Neural systems recruited by drug- and food-related cues: studies of gene activation in corticolimbic regions.

In order to survive, animals must acquire information about the reward value of stimuli in their environment. This process partly depends on the ability of the organism to make associations between the environmental context and the internal representation of value. While this type of learning probably evolved in order to promote behaviors that increase fitness (e.g., ingestive and sexual behavior), neuropsychological research utilizing addictive drugs, which are potent artificial reinforcers, has led to a deeper understanding of reinforcement mechanisms. Through these associations, sensory cues can acquire emotional salience and motivational properties. Exposure to drug-related cues in human addicts results in drug craving and localized activation of central circuits that are known to mediate cue-induced reinstatement of drug-seeking behavior in animal models of relapse. Similar regional activation patterns occur in humans in response to cues associated with foods. Furthermore, drug- and food-related cues not only activate common neuroanatomical regions but also result in similar activity-regulated gene expression programs within these shared areas. Here we discuss recent studies from our laboratory that investigate gene expression patterns elicited by exposure to palatable food- or drug-related cues. These studies suggest that the central nervous system stores and utilizes information about 'natural' and drug reinforcers in similar ways, both neuroanatomically and biochemically. These considerations may have important implications for the pharmacological and cognitive-behavioral treatments of substance use disorders, addiction, eating disorders, and obesity.

Vertebrate GLD2 poly(A) polymerases in the germline and the brain.

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.

Contextual cues associated with nicotine administration increase arc mRNA expression in corticolimbic areas of the rat brain.

Conditioned responses to cues associated with the administration of drugs of misuse are an impediment to continued abstinence for drug-free addicted individuals. In order to study the neuroanatomical and cellular response of the brain to cues associated with nicotine administration, we conditioned Sprague-Dawley rats to receive an ascending dose regimen of nicotine over 14 days in two distinct non-home cage environments and assessed expression of the early response gene arc in corticolimbic areas in response to the nicotine-associated context. All of the rats received the same dose regimen of nicotine. Three days after the last training day, the rats were exposed to the test environment. The rats that had previously received nicotine exhibited increased motor activity compared with the rats that had received saline in the test environment. After 45 min in the test environment, brains were taken for Northern blotting and in situ hybridization analysis, which revealed an increase in levels of activity-regulated, dendritically localized mRNA for arc in a variety of brain regions (medial and lateral prefrontal cortices, cingulate cortex, primary sensory cortex, sensorimotor cortex, ventral striatum and amygdala). Plasma corticosterone levels were not different between the groups, suggesting that exposure to nicotine cues is insufficient to activate the hypothalamo-pituitary-adrenal axis. Given that Arc plays a direct role in neuronal plasticity and memory consolidation, its induction by nicotine-associated cues in brain regions critical for cognitive and emotional processing suggests that rats may be learning that these cues are no longer necessarily predictive of nicotine administration. Further work will be needed in order to assess the role of arc expression in the extinction of conditioned responses to drug-paired cues.

Risk taking and novelty seeking in adolescence: introduction to part I.

Risk taking and novelty seeking are hallmarks of typical adolescent behavior. Adolescents seek new experiences and higher levels of rewarding stimulation, and often engage in risky behaviors, without considering future outcomes or consequences. These behaviors can have adaptive benefits with regard to the development of independence and survival without parental protection, but also render the adolescent more vulnerable to harm. Indeed, the risk of injury or death is higher during the adolescent period than in childhood or adulthood, and the incidence of depression, anxiety, drug use and addiction, and eating disorders increases. Brain pathways that play a key role in emotional regulation and cognitive function undergo distinct maturational changes during this transition period. It is clear that adolescents think and act differently from adults, yet relatively little is known about the precise mechanisms underlying neural, behavioral, and cognitive events during this period. Increased investigation of these dynamic alterations, particularly in prefrontal and related corticolimbic circuitry, may aid this understanding. Moreover, the investigation of mammalian animal models of adolescence-such as those examining impulsivity, reward sensitivity, and decision making-may also provide new opportunities for addressing the problem of adolescent vulnerability.

Comparison of hypocretin/orexin and melanin-concentrating hormone neurons and axonal projections in the embryonic and postnatal rat brain.

Hypocretin/orexin (H/O) and melanin-concentrating hormone (MCH) are peptide neuromodulators found in separate populations of neurons located within the lateral and perifornical hypothalamic regions. H/O has been linked to sleep-wakefulness regulation and to the sleep disorder narcolepsy, and both systems have been implicated in energy homeostasis, including the regulation of food intake. In the present study we compared the development of H/O and MCH-expressing neuronal populations with in situ hybridization and immunohistochemistry on adjacent sections in the embryonic and postnatal rat brain. We found that MCH mRNA and protein were present in developing neurons of the hypothalamus by embryonic day 16 (E16), whereas H/O mRNA and protein were not detected until E18. We also identified previously undescribed populations of MCH-immunoreactive cells in the lateral septum, paraventricular hypothalamic nucleus, lateral zona incerta, and ventral lateral geniculate nucleus that may play a specific role in the development of these regions. MCH immunoreactive axonal processes were also evident earlier than H/O stained fibers and at the time H/O immunoreactive processes were first identified in the hypothalamus at E20, extensive MCH axonal fiber systems were already present in many brain regions. Interestingly, however, the density of axonal fibers immunoreactive for H/O in the locus coeruleus reached peak levels at the same developmental age (P21) as MCH immunoreactive axons in the diagonal band of Broca (DBB). The peak of axon density coincided with the developmental stage at which adult patterns of feeding and sleep-waking activity become established. The present results demonstrate developmental differences and similarities between the MCH and H/O systems that may relate to their respective roles in feeding and sleep regulation.

Differential behavioral effects of nicotine exposure in adolescent and adult rats.

RATIONALE: Although the detrimental effects of nicotine in early brain development and the addictive properties in adulthood are well known, little is known about the neurobiological effects of nicotine in adolescence. An important question is whether adolescents and adults differ in the development of nicotine sensitization and drug-cue conditioning. OBJECTIVE: To examine the behavioral effects of multiple, repeated injections of nicotine on both sensitization and drug-cue conditioning in the adolescent rat, and to compare this profile with the adult rat. METHODS: Sixteen male adolescent (28 day) and 16 young adult (70 day) rats were given injections of either saline or nicotine and tested for motor activity for 90 min for ten consecutive days. Following 4 days of no testing, animals were given a sham injection and placed in the testing apparatus for 90 min. A dose-response curve for nicotine was also generated using two additional groups of ten adolescent and ten adult male rats. RESULTS: Adolescent rats, unlike adults, did not exhibit signs of nicotine-cue conditioning, and displayed less robust sensitization to the locomotor effects of nicotine than adults. Dose-response testing revealed differences in adolescent responsivity to nicotine in measures of rearing, but not ambulation. Initial exposure to nicotine resulted in increased sensitivity to the motor-activating effects of nicotine but less sensitivity to the depressant effects of nicotine in rearing in adolescents. CONCLUSIONS: Adolescent animals display different long-term neuroadaptive responses to nicotine than adult animals, possibly related to immature or still-developing plasticity mechanisms in the prefrontal cortex.

Activation of a subpopulation of orexin/hypocretin-containing hypothalamic neurons by GABAA receptor-mediated inhibition of the nucleus accumbens shell, but not by exposure to a novel environment.

Gamma-amino butyric acid (GABA)A receptor stimulation in the nucleus accumbens shell produces intense hyperphagia in rats and increases Fos expression in the lateral hypothalamus. To explore the involvement of hypothalamic orexin/hypocretin- or melanin concentrating hormone-immunoreactive neurons in this effect, the GABAA agonist, muscimol (0, 50 ng), was infused directly into the nucleus accumbens shell of rats; 90 min later, their brains were collected and subsequently processed for immunohistochemistry. A group exposed to a novel environment was included to evaluate the specificity of Fos expression changes with regard to general arousal. Alternating sections through the hypothalamus were double-stained for orexin/hypocretin-Fos or melanin concentrating hormone-Fos combinations. Intra-accumbens shell muscimol treatment significantly increased the percentage of orexin/hypocretin-containing neurons expressing Fos in the lateral, but not medial, portion of the perifornical/lateral hypothalamic area. Regardless of treatment condition, greater percentages of orexin/hypocretin-containing neurons in the medial portion of the hypothalamus expressed Fos relative to cells located more laterally. None of the manipulations increased Fos expression in melanin concentrating hormone-immunoreactive neurons. Muscimol treatment also markedly increased Fos expression in the arcuate nucleus, which connects reciprocally to the lateral/perifornical hypothalamic area. Thus, orexin/hypocretin-containing neurons in lateral sectors of the hypothalamus, along with cells in the arcuate nucleus, display phasic increases in Fos expression after an orexigenic pharmacological manipulation of the nucleus accumbens shell, but to a lesser degree after the heightened arousal associated with exposure to a novel environment.