Fornix volumetric increase and microglia morphology contribute to spatial and recognition-like memory decline in ageing male mice.

Ageing displays a low-grade pro-inflammatory profile in blood and the brain. Accumulation of pro-inflammatory cytokines, microglia activation and volumetric changes in the brain correlate with cognitive decline in ageing models. However, the interplay between them is not totally understood. Here, we aimed to globally identify an age-dependent pro-inflammatory profile and microglia morphological plasticity that favors major volume changes in the brain associated with cognitive decline. Cluster analysis of behavioral data obtained from 2-,12- and 20-month-old male C57BL/6 mice revealed age-dependent cognitive decline after the Y-maze, Barnes maze, object recognition (NORT) and object location tests (OLT). Global magnetic resonance imageing (MRI) analysis by deformation-based morphometry (DBM) in the brain identified a volume increase in the fornix and a decrease in the left medial entorhinal cortex (MEntC) during ageing. Notably, the fornix shows an increase in the accumulation of pro-inflammatory cytokines, whereas the left MEntC displays a decrease. Morphological assessment of microglia also confirms an active and dystrophic phenotype in the fornix and a surveillance phenotype in the left MEntC. Finally, biological modeling revealed that age-related volume increase in the fornix was associated with dystrophic microglia and cognitive impairment, as evidenced by failure on tasks examining memory of object location and novelty. Our results propose that the morphological plasticity of microglia might contribute to volumetric changes in brain regions associated with cognitive decline during physiological ageing.

Soft Drink Consumption in Young Mexican Adults Is Associated with Higher Total Body Fat Percentage in Men but Not in Women.

A high consumption of soft drinks (SDs) has been linked with the development of anthropometric and metabolic alterations. We evaluate the association between SD consumption and some anthropometric and metabolic variables. This study is an observational study, using a sample of 394 university students, of which 158 were men (40.1%) and 238 women (59.9%), between 18 and 30 years. An SD intake questionnaire provided the consumption of different SDs. The participants' weight, height, and waist and hip circumferences were collected. Metabolic biomarkers were analyzed. The average intake of caloric SDs (CSDs) was 1193.6 ± 1534.8 mL/week and 84.5 ± 115.02 mL/week for non-caloric SDs (NCSDs). Sex differences were found in the amount of SD consumption and these statistical differences were driven by those men subjects with a high total body fat percentage (TBF%). In men, correlations were found between the intake of CSDs and the body mass index, waist and hip circumferences, TBF%, and visceral fat percentage. In woman, a correlation was found with glucose and triglycerides. The prediction model revealed that the intake of CSDs predicts TBF% and low-density lipoprotein only in men. A high amount of CSD consumption in men was associated with a high TBF%, and this may be predictive of future development of metabolic abnormalities.

Morris water maze overtraining increases the density of thorny excrescences in the basal dendrites of CA3 pyramidal neurons.

The hippocampus plays a fundamental role in spatial learning and memory. Dentate gyrus (DG) granular neurons project mainly to proximal apical dendrites of neurons in the CA3 stratum lucidum and also, to some extent, to the basal dendrites of CA3 pyramidal cells in the stratum oriens. The terminal specializations of DG neurons are the mossy fibers (MF), and these huge axon terminals show expansion in the CA3 stratum oriens after the animals undergo overtraining in the Morris Water Maze task (MWM). However, to our knowledge there are no reports regarding the possible changes in density of post-synaptic targets of these terminals in the basal dendrites of CA3 neurons after overtraining in the MWM. The purpose of this work was to study the density of thorny excrescences (TE) and other dendritic spine types (stubby, thin, and mushroom) in the CA3 stratum oriens in animals overtrained in the MWM for three consecutive days and in animals trained for only one day. Seven days after MWM training, the animals were sacrificed, and their brains removed and processed for rapid Golgi staining to visualize the different types of dendritic protrusions. Our results revealed that the relative quantity of stubby, thin, and mushroom dendritic spines did not change, regardless of amount of training. However, a significant increase in the density of TE was detected in the overtrained animals. These results strongly suggest that spatial water maze overtraining induces an increased density of MF-TE connections, which might be functionally relevant for long-term spatial memory formation.

Los factores de la teoría de la conducta planeada relacionados con el patrón de consumo de bebidas endulzadas en jóvenes universitarios / Factors from the theory of planned behavior and soda consumption patterns among university students

RESUMEN En México, el consumo de bebidas endulzadas o refrescos es uno de los más altos a nivel mundial, esto representa un factor de riesgo para el desarrollo de obesidad y enfermedades metabólicas. En este estudio empleamos la Teoría de la Conducta Planeada (TCP) para identificar factores cognitivos asociados a la frecuencia de consumo de refrescos en adultos jóvenes mexicanos. Estudiamos una muestra de 261 personas, 110 hombres y 151 mujeres, de 18 a 45 años de edad (M= 22.51; DE = 4.2). Los participantes completaron cuestionarios de frecuencia de consumo e instrumentos basados en la TCP. Se encontraron dos factores predictores independientes y significativos de la frecuencia de consumo, el principal fue la baja autoeficacia, seguido por la intención. Ambos factores explican el 27.3% de la varianza de la frecuencia de consumo. Estos resultados contribuyen a entender los factores cognitivos involucrados en el consumo de refrescos, y sugieren que la alta frecuencia de consumo es una conducta no racional, por lo que podrían estar implicados aspectos afectivos y motivacionales, lo que deberá ser dilucidado con más investigación que permita explorar nuevas alternativas para el desarrollo de programas de prevención y tratamiento del consumo de bebidas con riesgo para la salud humana.

ABSTRACT Consumption of sugar sweetened beverages (SSB) in Mexico is one of the highest in the world, which represents a risk factor for the development of obesity and metabolic diseases. In the present study we used the Theory of Planned Behavior (TPB) to identify cognitive factors associated with the frequency of SSB consumption in Mexican young adults. We studied a sample of 261 people, 110 men and 151 women, from 18 to 45 years of age (M = 22.51, SD = 4.2). Participants completed consumption frequency questionnaires and instruments based on TPB. Two independent and significant predictors of consumption frequency were found: low self-efficacy and intention. Both factors explained 27.3% of the variance in consumption frequency. These results contribute to the understanding of the cognitive factors underlying SSB consumption, and suggest that high consumption frequency is a non-rational behaviour. It is possible that affective and motivational aspects could also be involved, which should be clarified with more research, in order to explore alternatives for the prevention and treatment of unhealthy consumption of beverages that are a risk to human health.

Evidence for an Evolutionarily Conserved Memory Coding Scheme in the Mammalian Hippocampus.

Decades of research identify the hippocampal formation as central to memory storage and recall. Events are stored via distributed population codes, the parameters of which (e.g., sparsity and overlap) determine both storage capacity and fidelity. However, it remains unclear whether the parameters governing information storage are similar between species. Because episodic memories are rooted in the space in which they are experienced, the hippocampal response to navigation is often used as a proxy to study memory. Critically, recent studies in rodents that mimic the conditions typical of navigation studies in humans and nonhuman primates (i.e., virtual reality) show that reduced sensory input alters hippocampal representations of space. The goal of this study was to quantify this effect and determine whether there are commonalities in information storage across species. Using functional molecular imaging, we observe that navigation in virtual environments elicits activity in fewer CA1 neurons relative to real-world conditions. Conversely, comparable neuronal activity is observed in hippocampus region CA3 and the dentate gyrus under both conditions. Surprisingly, we also find evidence that the absolute number of neurons used to represent an experience is relatively stable between nonhuman primates and rodents. We propose that this convergence reflects an optimal ensemble size for episodic memories.SIGNIFICANCE STATEMENT One primary factor constraining memory capacity is the sparsity of the engram, the proportion of neurons that encode a single experience. Investigating sparsity in humans is hampered by the lack of single-cell resolution and differences in behavioral protocols. Sparsity can be quantified in freely moving rodents, but extrapolating these data to humans assumes that information storage is comparable across species and is robust to restraint-induced reduction in sensory input. Here, we test these assumptions and show that species differences in brain size build memory capacity without altering the structure of the data being stored. Furthermore, sparsity in most of the hippocampus is resilient to reduced sensory information. This information is vital to integrating animal data with human imaging navigation studies.

Differential Arc protein expression in dorsal and ventral striatum after moderate and intense inhibitory avoidance training.

Intense training refers to training mediated by emotionally arousing experiences, such as aversive conditioning motivated by relatively high intensities of foot-shock, which produces a strong memory that is highly resistant to extinction. Intense training protects memory consolidation against the amnestic effects of a wide variety of treatments, administered systemically or directly into brain structures. The mechanisms of this protective effect are unknown. To determine a potential neurobiological correlate of the protective effect of intense training, rats were trained in a one-trial step-through inhibitory avoidance task using different intensities of foot-shock (0.0, 0.5, 1.0, and 2.0mA). Some rats from each group were sacrificed 45min after training for immunohistochemical Arc protein detection in dorsal and ventral striatum; other rats were tested for extinction during six consecutive days, starting 48h after training. The results showed that training with 1.0 and 2.0mA produced optimal retention scores, which were significantly higher than those of the 0.5 and 0.0mA groups. Also, a higher resistance to extinction was obtained with 2.0mA than with the other intensities. A high number of neurons expressed Arc in ventral, but not in dorsal striatum in both the 1.0 and 2.0mA groups, with a larger area of Arc signal in the latter group. We conclude that an increased Arc expression may be related to enhanced synaptic plasticity in the ventral striatum, suggesting that it may be one of the physiological substrates of enhanced learning.

Hippocampal Synaptic Expansion Induced by Spatial Experience in Rats Correlates with Improved Information Processing in the Hippocampus.

Spatial water maze (WM) overtraining induces hippocampal mossy fiber (MF) expansion, and it has been suggested that spatial pattern separation depends on the MF pathway. We hypothesized that WM experience inducing MF expansion in rats would improve spatial pattern separation in the hippocampal network. We first tested this by using the the delayed non-matching to place task (DNMP), in animals that had been previously trained on the water maze (WM) and found that these animals, as well as animals treated as swim controls (SC), performed better than home cage control animals the DNMP task. The "catFISH" imaging method provided neurophysiological evidence that hippocampal pattern separation improved in animals treated as SC, and this improvement was even clearer in animals that experienced the WM training. Moreover, these behavioral treatments also enhance network reliability and improve partial pattern separation in CA1 and pattern completion in CA3. By measuring the area occupied by synaptophysin staining in both the stratum oriens and the stratun lucidum of the distal CA3, we found evidence of structural synaptic plasticity that likely includes MF expansion. Finally, the measures of hippocampal network coding obtained with catFISH correlate significantly with the increased density of synaptophysin staining, strongly suggesting that structural synaptic plasticity in the hippocampus induced by the WM and SC experience is related to the improvement of spatial information processing in the hippocampus.

Membrane biofouling mechanism in an aerobic granular reactor degrading 4-chlorophenol.

The membrane fouling of an aerobic granular reactor coupled with a submerged membrane in a sequencing batch reactor (SBR) was evaluated. The fouling analysis was performed by applying microscopy techniques to determine the morphology and structure of the fouling layer on a polyvinylidene fluoride membrane. It was found that the main cause of fouling was the polysaccharide adsorption on the membrane surface, followed by the growth of microorganisms to form a biofilm.

Differential effects of spaced vs. massed training in long-term object-identity and object-location recognition memory.

Here we tested whether the well-known superiority of spaced training over massed training is equally evident in both object identity and object location recognition memory. We trained animals with objects placed in a variable or in a fixed location to produce a location-independent object identity memory or a location-dependent object representation. The training consisted of 5 trials that occurred either on one day (Massed) or over the course of 5 consecutive days (Spaced). The memory test was done in independent groups of animals either 24h or 7 days after the last training trial. In each test the animals were exposed to either a novel object, when trained with the objects in variable locations, or to a familiar object in a novel location, when trained with objects in fixed locations. The difference in time spent exploring the changed versus the familiar objects was used as a measure of recognition memory. For the object-identity-trained animals, spaced training produced clear evidence of recognition memory after both 24h and 7 days, but massed-training animals showed it only after 24h. In contrast, for the object-location-trained animals, recognition memory was evident after both retention intervals and with both training procedures. When objects were placed in variable locations for the two types of training and the test was done with a brand-new location, only the spaced-training animals showed recognition at 24h, but surprisingly, after 7 days, animals trained using both procedures were able to recognize the change, suggesting a post-training consolidation process. We suggest that the two training procedures trigger different neural mechanisms that may differ in the two segregated streams that process object information and that may consolidate differently.

Sustained transcription of the immediate early gene Arc in the dentate gyrus after spatial exploration.

After spatial exploration in rats, Arc mRNA is expressed in ∼2% of dentate gyrus (DG) granule cells, and this proportion of Arc-positive neurons remains stable for ∼8 h. This long-term presence of Arc mRNA following behavior is not observed in hippocampal CA1 pyramidal cells. We report here that in rats ∼50% of granule cells with cytoplasmic Arc mRNA, induced some hours previously during exploration, also show Arc expression in the nucleus. This suggests that recent transcription can occur long after the exploration behavior that elicited it. To confirm that the delayed nuclear Arc expression was indeed recent transcription, Actinomycin D was administered immediately after exploration. This treatment resulted in inhibition of recent Arc expression both when evaluated shortly after exploratory behavior as well as after longer time intervals. Together, these data demonstrate a unique kinetic profile for Arc transcription in hippocampal granule neurons following behavior that is not observed in other cell types. Among a number of possibilities, this sustained transcription may provide a mechanism that ensures that the synaptic connection weights in the sparse population of granule cells recruited during a given behavioral event are able to be modified.

Network, cellular, and molecular mechanisms underlying long-term memory formation.

The neural network stores information through activity-dependent synaptic plasticity that occurs in populations of neurons. Persistent forms of synaptic plasticity may account for long-term memory storage, and the most salient forms are the changes in the structure of synapses. The theory proposes that encoding should use a sparse code and evidence suggests that this can be achieved through offline reactivation or by sparse initial recruitment of the network units. This idea implies that in some cases the neurons that underwent structural synaptic plasticity might be a subpopulation of those originally recruited; However, it is not yet clear whether all the neurons recruited during acquisition are the ones that underwent persistent forms of synaptic plasticity and responsible for memory retrieval. To determine which neural units underlie long-term memory storage, we need to characterize which are the persistent forms of synaptic plasticity occurring in these neural ensembles and the best hints so far are the molecular signals underlying structural modifications of the synapses. Structural synaptic plasticity can be achieved by the activity of various signal transduction pathways, including the NMDA-CaMKII and ACh-MAPK. These pathways converge with the Rho family of GTPases and the consequent ERK 1/2 activation, which regulates multiple cellular functions such as protein translation, protein trafficking, and gene transcription. The most detailed explanation may come from models that allow us to determine the contribution of each piece of this fascinating puzzle that is the neuron and the neural network.

Neurons generated in senescence maintain capacity for functional integration.

Adult-born neurons in the dentate gyrus (DG) can survive for long periods, are capable of integrating into neuronal networks, and are important for hippocampus-dependent learning. Neurogenesis is dramatically reduced during senescence, and it remains unknown whether those few neurons that are produced remain capable of network integration. The expression of Arc, a protein coupled to neuronal activity, was used to measure activity among granule cells that were labeled with BrdU 4 months earlier in young (9 months) and aged (25 months) Fischer344 rats. The results indicate that while fewer cells are generated in the senescent DG, those that survive are (a) more likely to respond to spatial processing by expressing Arc relative to the remainder of the granule cell population and (b) equally responsive to spatial exploration as granule cells of the same age from young animals. These findings provide compelling evidence that newborn granule cells in the aged DG retain the capacity for participation in functional hippocampal networks.

Familiar taste induces higher dendritic levels of activity-regulated cytoskeleton-associated protein in the insular cortex than a novel one.

The immediate early gene (IEG) Arc is known to play an important role in synaptic plasticity; its protein is locally translated in the dendrites where it has been involved in several types of plasticity mechanisms. Because of its tight coupling with neuronal activity, Arc has been widely used as a tool to tag behaviorally activated networks. However, studies examining the modulation of Arc expression during and after learning have yielded somewhat contradictory results. Although some have reported that higher levels of Arc were induced by initial acquisition of a task rather than by reinstating a learned behavior, others have failed to observe such habituation of Arc transcription. Moreover, most of these studies have focused on the mRNA and, surprisingly, relatively little is known about how learning can affect Arc protein expression levels. Here we used taste recognition memory and examined Arc protein expression in the insular cortex of rats at distinct times during taste memory formation. Interestingly, we found that more Arc protein was induced by a familiar rather than by a novel taste. Moreover, this increase was inhibited by post-trial intrahippocampal anisomycin injections, a treatment known to inhibit safe-taste memory consolidation. In addition, confocal microscopy analysis of immunofluorescence stained tissue revealed that the proportion of IC neurons expressing Arc was the same in animals exposed to novel and familiar taste, but Arc immunoreactivity in dendrites was dramatically higher in rats exposed to the familiar taste. These results provide novel insights on how experience affects cortical plasticity.

When are new hippocampal neurons, born in the adult brain, integrated into the network that processes spatial information?

Adult-born neurons in the dentate gyrus (DG) functionally integrate into the behaviorally relevant hippocampal networks, showing a specific Arc-expression response to spatial exploration when mature. However, it is not clear when, during the 4- to 6-week interval that is critical for survival and maturation of these neurons, this specific response develops. Therefore, we characterized Arc expression after spatial exploration or cage control conditions in adult-born neurons from rats that were injected with BrdU on one day and were sacrificed 1, 7, 15, 30, and 45 days post-BrdU injection (PBI). Triple immunostaining for NeuN, Arc, and BrdU was analyzed through the different DG layers. Arc protein expression in BrdU-positive cells was observed from day 1 to day 15 PBI but was not related to behavioral stimulation. The specific Arc-expression response to spatial exploration was observed from day 30 and 45 in about 5% of the BrdU-positive cell population. Most of the BrdU-positive neurons expressing Arc in response to spatial exploration (∼90%) were located in DG layer 1, and no Arc expression was observed in cells located in the subgranular zone (SGZ). Using the current data and that obtained previously, we propose a mathematical model suggesting that new neurons are unlikely to respond to exploration by expressing Arc after they are 301 days old, and also that in a 7-month-old rat the majority (60%) of the neurons that respond to exploration must have been born during adulthood; thus, suggesting that adult neurogenesis in the DG is highly relevant for spatial information processing.

Flavor preference learning increases olfactory and gustatory convergence onto single neurons in the basolateral amygdala but not in the insular cortex in rats.

The basolateral amygdala (BLA) and the insular cortex (IC) represent two major areas for odor-taste associations, i.e. flavor integration. This learning may require the development of convergent odor and taste neuronal activation allowing the memory representation of such association. Yet identification of neurons that respond to such coincident input and the effect of flavor experience on odor-taste convergence remain unclear. In the present study we used the compartmental analysis of temporal activity using fluorescence in situ hybridization for Arc (catFISH) to visualize odor-taste convergence onto single neurons in the BLA and in the IC to assess the number of cells that were co-activated by both stimuli after odor-taste association. We used a sucrose conditioned odor preference as a flavor experience in rats, in which 9 odor-sucrose pairings induce a reliable odor-taste association. The results show that flavor experience induced a four-fold increase in the percentage of cells activated by both taste and odor stimulations in the BLA, but not in the IC. Because conditioned odor preference did not modify the number of cells responding selectively to one stimulus, this greater odor-taste convergence into individual BLA neurons suggests the recruitment of a neuronal population that can be activated by both odor and taste only after the association. We conclude that the development of convergent activation in amygdala neurons after odor-taste associative learning may provide a cellular basis of flavor memory.

The expression of TRH, its receptors and degrading enzyme is differentially modulated in the rat limbic system during training in the Morris water maze.

TRH administration induces arousal, improves cognition, and modulates glutamatergic and cholinergic transmission in hippocampal neurons. To study the possible involvement of TRH neurons in learning and memory processes, gene expression of TRH, its receptors, and pyroglutamyl peptidase (PPII), were measured in limbic regions of water-maze trained rats. Hypothalamus and amygdala showed changes related to the task but not specific to spatial learning while in hippocampus, pro-TRH and TRH-R1 mRNA levels were specifically increased in those animals trained to find a hidden platform. Variation of TRH content and mRNA levels of pro-TRH, TRH-R1, TRH-R2 and PPII are observed in conditions known to activate TRH hypophysiotropic neurons. Changes in some of these parameters could indicate the activation of TRHergic neurons and their possible involvement in some memory related process. Male Wistar rats were immersed (10 times) for 1, 3 or 5 days in a Morris water-maze containing, or not (yoked control) a platform and sacrificed 5, 30 and 60 min after last trial. TRH content and TSH serum levels were determined by radioimmunoassay; mRNA levels of pro-TRH, TRH-R1, TRH-R2, and PPII, by RT-PCR. Exclusive changes due to spatial training were observed in posterior hippocampus of rats trained for 5 days sacrificed after 60min: decreased TRH content and increased mRNA levels of pro-TRH and TRH-R1, particularly in CA3 region (measured by in situ hybridization). The hypothalamus-pituitary axis responded in both yoked and trained animals (increasing serum TSH levels and pro-TRH expression, due to swim-stress); in the amygdala of both groups, pro-TRH expression increased while diminished that of both receptors and PPII. Differential expression of these parameters suggests involvement of TRH hippocampal neurons in memory formation processes while changes in amygdala could relate to TRH anxiolytic role. The differential modulation in anterior and posterior portions of the hippocampus is discussed.

Integration of new neurons into functional neural networks.

Although it is established that new granule cells can be born and can survive in the adult mammalian hippocampus, there remains some question concerning the functional integration of these neurons into behaviorally relevant neural networks. By using high-resolution confocal microscopy, we have applied a new strategy to address the question of functional integration of newborn neurons into networks that mediate spatial information processing and memory formation. Exploration-induced expression of the immediate-early gene Arc in hippocampal cells has been linked to cellular activity observed in electrophysiological recordings under the same behavioral conditions. We investigated whether mature (5-month-old), newborn granule cells express Arc in response to a discrete spatial experience by detecting the expression of Arc in combination with NeuN (neuron-specific nuclear protein)-positive and bromodeoxyuridine-positive cells. We found that mature new granule cells do indeed express Arc in response to an exploration experience, supporting the idea that these cells are well integrated into hippocampal circuits. The proportion of mature newborn neurons that expressed Arc in response to exploration, however, was significantly higher (approximately 2.8%) than the proportion of cells that expressed Arc in the already existing population of granule cells (approximately 1.6%; p < 0.01). This finding extends previous data suggesting that the cellular physiology of newborn granule neurons differs from that of the existing population by indicating that these properties are retained in mature adult-generated neurons. Thus, these data have interesting implications for network models of spatial information processing and the role of hippocampal circuits in memory, indicating that mature new neurons are selectively recruited into hippocampal cell assemblies in higher proportions than older cells.

Spatial exploration induces ARC, a plasticity-related immediate-early gene, only in calcium/calmodulin-dependent protein kinase II-positive principal excitatory and inhibitory neurons of the rat forebrain.

Active behavior, such as exploring a novel environment, induces the expression of the immediate-early gene Arc (activity-regulated cytoskeletal associated protein, or Arg 3.1) in many brain regions, including the hippocampus, neocortex, and striatum. Arc messenger ribonucleic acid and protein are localized in activated dendrites, and Arc protein is required for the maintenance of long-term potentiation and memory consolidation. Although previous evidence suggests that Arc is expressed in neurons, there is no direct demonstration that only neurons can express Arc. Furthermore, there is no characterization of the main neuronal types that express Arc. The data reported here show that behavior- or seizure-induced Arc expression in the hippocampus, primary somatosensory cortex, and dorsal striatum of rats colocalizes only with neuronal (NeuN-positive) and not with glial (GFAP-positive) cells. Furthermore, Arc was found exclusively in non-GABAergic alpha-CaMKII-positive hippocampal and neocortical neurons of rats that had explored a novel environment. Some GAD65/67-positive neurons in these regions were observed to express Arc, but only after a very strong stimulus (electroconvulsive seizure). In the dorsal striatum, spatial exploration induced Arc only in GABAergic and alpha-CaMKII-positive neurons. Combined, these results show that although a very strong stimulus (seizure) can induce Arc in a variety of neurons, behavior induces Arc in the CaMKII-positive principal neurons of the hippocampus, neocortex, and dorsal striatum. These results, coupled with recent in vitro findings of interactions between Arc and CaMKII, are consistent with the hypothesis that Arc and CaMKII act as plasticity partners to promote functional and/or structural synaptic modifications that accompany learning.