Enduring effects of an unhealthy diet during adolescence on systemic but not neurobehavioural measures in adult rats.

Introduction: Adolescence is an important stage of maturation for various brain structures. It is during this time therefore that the brain may be more vulnerable to environmental factors such as diet that may influence mood and memory. Diets high in fat and sugar (termed a cafeteria diet) during adolescence have been shown to negatively impact upon cognitive performance, which may be reversed by switching to a standard diet during adulthood. Consumption of a cafeteria diet increases both peripheral and central levels of interleukin-1ß (IL-1ß), a pro-inflammatory cytokine which is also implicated in cognitive impairment during the ageing process. It is unknown whether adolescent exposure to a cafeteria diet potentiates the negative effects of IL-1ß on cognitive function during adulthood.Methods: Male Sprague-Dawley rats consumed a cafeteria diet during adolescence after which time they received a lentivirus injection in the hippocampus to induce chronic low-grade overexpression of IL-1ß. After viral integration, metabolic parameters, circulating and central pro-inflammatory cytokine levels, and cognitive behaviours were assessed.Results: Our data demonstrate that rats fed the cafeteria diet exhibit metabolic dysregulations in adulthood, which were concomitant with low-grade peripheral and central inflammation. Overexpression of hippocampal IL-1ß in adulthood impaired spatial working memory. However, adolescent exposure to a cafeteria diet, combined with or without hippocampal IL-1ß in adulthood did not induce any lasting cognitive deficits when the diet was replaced with a standard diet in adulthood. Discussion: These data demonstrate that cafeteria diet consumption during adolescence induces metabolic and inflammatory changes, but not behavioural changes in adulthood.

Lactobacillus rhamnosus GG soluble mediators ameliorate early life stress-induced visceral hypersensitivity and changes in spinal cord gene expression.

Visceral hypersensitivity is a hallmark of many functional and stress-related gastrointestinal disorders, and there is growing evidence that the gut microbiota may play a role in its pathophysiology. It has previously been shown that early life stress-induced visceral sensitivity is reduced by various probiotic strains of bacteria (including Lactobacillus rhamnosus GG (LGG)) alone or in combination with prebiotic fibres in rat models. However, the exact mechanisms underpinning such effects remain unresolved. Here, we investigated if soluble mediators derived from LGG can mimic the bacteria's effects on visceral hypersensitivity and the microbiota-gut-brain axis. Rats were exposed to maternal separation (MS) from postnatal days 2-12. From weaning onwards both non-separated (NS) and MS offspring were provided drinking water with or without supplementation of standardized preparations of the LGG soluble mediators (LSM). Our results show that MS led to increased visceral sensitivity and exaggerated corticosterone plasma levels following restraint stress in adulthood, and both of these effects were ameliorated through LSM supplementation. Differential regulation of various genes in the spinal cord of MS versus NS rats was observed, 41 of which were reversed by LSM supplementation. At the microbiota composition level MS led to changes in beta diversity and abundance of specific bacteria including parabacteroides, which were ameliorated by LSM. These findings support probiotic soluble mediators as potential interventions in the reduction of symptoms of visceral hypersensitivity.

Enduring Behavioral Effects Induced by Birth by Caesarean Section in the Mouse.

Birth by Caesarean (C)-section impacts early gut microbiota colonization and is associated with an increased risk of developing immune and metabolic disorders. Moreover, alterations of the microbiome have been shown to affect neurodevelopmental trajectories. However, the long-term effects of C-section on neurobehavioral processes remain unknown. Here, we demonstrated that birth by C-section results in marked but transient changes in microbiome composition in the mouse, in particular, the abundance of Bifidobacterium spp. was depleted in early life. Mice born by C-section had enduring social, cognitive, and anxiety deficits in early life and adulthood. Interestingly, we found that these specific behavioral alterations induced by the mode of birth were also partially corrected by co-housing with vaginally born mice. Finally, we showed that supplementation from birth with a Bifidobacterium breve strain, or with a dietary prebiotic mixture that stimulates the growth of bifidobacteria, reverses selective behavioral alterations in C-section mice. Taken together, our data link the gut microbiota to behavioral alterations in C-section-born mice and suggest the possibility of developing adjunctive microbiota-targeted therapies that may help to avert long-term negative consequences on behavior associated with C-section birth mode.

Host Microbiota Regulates Central Nervous System Serotonin Receptor 2C Editing in Rodents.

Microbial colonization of the gastrointestinal tract plays a crucial role in the development of enteric and central nervous system functionality. The serotonergic system has been heavily implicated in microbiota-gut-brain axis signaling, particularly in proof-of-principle studies in germ-free (GF) animals. One aspect of the serotonergic system that has been left unexplored in relation to the microbiota is the unique ability of the serotonin receptor 2C (5-HT2C) to undergo post-transcriptional editing, which has been implicated in decreased receptor functionality. We investigated whether GF mice, with absent microbiota from birth, have altered 5-HT2C receptor expression and editing in the brain, and if colonization of the microbiota is able to restore editing patterns. Next, we investigated whether microbiota depletion later in life using a chronic antibiotic treatment could affect 5-HT2C receptor editing patterns in rats. We found that GF mice have an increased prevalence of the edited 5-HT2C receptor isoforms in the amygdala, hypothalamus, prefrontal cortex, and striatum, which was partially normalized upon colonization post-weaning. However, no alterations were observed in the hypothalamus after microbiota depletion using an antibiotic treatment in adult rats. This suggests that alterations in the microbiome during development, but not later in life, could influence 5-HT2C receptor editing patterns. Overall, these results demonstrate that the microbiota affects 5-HT2C receptor editing in the brain and may inform novel therapeutic strategies in conditions in which 5-HT2C receptor editing is altered, such as depression.

Differential effects of adolescent and adult-initiated exercise on cognition and hippocampal neurogenesis.

Adolescence is a critical period for postnatal brain maturation and thus a time when environmental influences may affect cognitive processes in later life. Exercise during adulthood has been shown to increase hippocampal neurogenesis and enhance cognition. However, the impact of exercise initiated in adolescence on the brain and behavior in adulthood is not fully understood. The aim of this study was to compare the impact of voluntary exercise that is initiated during adolescence or early adulthood on cognitive performance in hippocampal-dependent and -independent processes using both object-based and touchscreen operant paradigms. Adult (8 week) and adolescent (4 week) male Sprague-Dawley rats had access to a running wheel (exercise) or were left undisturbed (sedentary control) for 4 weeks prior to behavioral testing and for the duration of the experiment. Results from touchscreen-based tasks showed that reversal learning was enhanced by both adult and adolescent-initiated exercise, while only exercise that began in adolescence induced a subtle but transient increase in performance on a location discrimination task. Spontaneous alternation in the Y-maze was impaired following adolescent onset exercise, while object memory was unaffected by either adult or adolescent-initiated exercise. Adolescent-initiated exercise increased the number of hippocampal DCX cells, an indicator of neurogenesis. It also promoted the complexity of neurites on DCX cells, a key process for synaptic integration, to a greater degree than adult-initiated exercise. Together the data here show that exercise during the adolescent period compared to adulthood differentially affects cognitive processes and the development of new hippocampal neurons in later life.

Neurobehavioural effects of Lactobacillus rhamnosus GG alone and in combination with prebiotics polydextrose and galactooligosaccharide in male rats exposed to early-life stress.

Early life is a period of significant brain development when the brain is at its most plastic and vulnerable. Stressful episodes during this window of development have long-lasting effects on the central nervous system. Rodent maternal separation (MS) is a reliable model of early-life stress and induces alterations in both physiology and behaviour. Intriguingly, the gut microbiota of MS offspring differ from that of non-separated offspring, suggesting a mechanistic role for the microbiota-gut-brain axis. Hence, we tested whether dietary factors known to affect the gut microbiota alter the neurobehavioural effects of MS. The impact of consuming diet containing prebiotics polydextrose (PDX) and galactooligosaccharide (GOS) alone or in combination with live bacteria Lactobacillus rhamnosus GG (LGG) from weaning onwards in rats subjected to early-life MS was assessed. Adult offspring were assessed for anxiety-like behaviour in the open field test, spatial memory using the Morris water maze, and reactivity to restraint stress. Brains were examined via PCR for changes in mRNA gene expression. Here, we demonstrate that diets containing a combination of PDX/GOS and LGG attenuates the effects of early-life MS on anxiety-like behaviour and hippocampal-dependent learning with changes to hippocampal mRNA expression of genes related to stress circuitry, anxiety and learning.

Differential effects of adolescent and adult-initiated voluntary exercise on context and cued fear conditioning.

Adolescence is a critical period for postnatal brain maturation and a time during which there is increased susceptibility to developing emotional and cognitive-related disorders. Exercise during adulthood has been shown to increase hippocampal plasticity and enhance cognition. However, the impact of exercise initiated in adolescence, on brain and behaviour in adulthood is not yet fully explored or understood. The aim of this study was to compare the impact of voluntary exercise that was initiated either during adolescence or early adulthood on cognitive performance in hippocampal and amygdala-dependent fear conditioning tasks in adulthood. Adult (eight weeks old) and adolescent (four weeks old) male Sprague Dawley rats had access to a running wheel (exercise) or were left undisturbed (sedentary control) for seven weeks. Adult-initiated exercise enhanced both contextual and cued fear conditioning, while conversely, exercise that began in adolescence did not affect performance in these tasks. These behaviours were accompanied by differential expression of plasticity-related genes in the hippocampus and amygdala in adulthood. Specifically, adolescent-initiated exercise increased the expression of an array of plasticity related genes in the hippocampus including BDNF, synaptophysin, Creb, PSD-95, Arc, TLX and DCX, while adult-initiated exercise did not affect hippocampal plasticity related genes. Together results show that exercise initiated during adolescence has a differential effect on hippocampal and amygdala-dependent behaviour and neuronal plasticity compared to when exercise was initiated in adulthood. These findings reinforce adolescence as a period during which environmental influences have a distinct impact on neuronal plasticity and cognition. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment".

A role for the orphan nuclear receptor TLX in the interaction between neural precursor cells and microglia.

Microglia are an essential component of the neurogenic niche in the adult hippocampus and are involved in the control of neural precursor cell (NPC) proliferation, differentiation and the survival and integration of newborn neurons in hippocampal circuitry. Microglial and neuronal cross-talk is mediated in part by the chemokine fractalkine/chemokine (C-X3-C motif) ligand 1 (CX3CL1) released from neurons, and its receptor CX3C chemokine receptor 1 (CX3CR1) which is expressed on microglia. A disruption in this pathway has been associated with impaired neurogenesis yet the specific molecular mechanisms by which this interaction occurs remain unclear. The orphan nuclear receptor TLX (Nr2e1; homologue of the Drosophila tailless gene) is a key regulator of hippocampal neurogenesis, and we have shown that in its absence microglia exhibit a pro-inflammatory activation phenotype. However, it is unclear whether a disturbance in CX3CL1/CX3CR1 communication mediates an impairment in TLX-related pathways which may have subsequent effects on neurogenesis. To this end, we assessed miRNA expression of up- and down-stream signalling molecules of TLX in the hippocampus of mice lacking CX3CR1. Our results demonstrate that a lack of CX3CR1 is associated with altered expression of TLX and its downstream targets in the hippocampus without significantly affecting upstream regulators of TLX. Thus, TLX may be a potential participant in neural stem cell (NSC)-microglial cross-talk and may be an important target in understanding inflammatory-associated impairments in neurogenesis.

Chronic interleukin-1ß in the dorsal hippocampus impairs behavioural pattern separation.

Understanding the long-term consequences of chronic inflammation in the hippocampus may help to develop therapeutic targets for the treatment of cognitive disorders related to stress, ageing and neurodegeneration. The hippocampus is particularly vulnerable to increases in the pro-inflammatory cytokine interleukin-1ß (IL-1ß), a mediator of neuroinflammation, with elevated levels implicated in the aetiology of neurodegenerative diseases such as Alzheimer's and Parkinson's, and in stress-related disorders such as depression. Acute increases in hippocampal IL-1ß have been shown to impair cognition and reduce adult hippocampal neurogenesis, the birth of new neurons. However, the impact of prolonged increases in IL-1ß, as evident in clinical conditions, on cognition has not been fully explored. Therefore, the present study utilized a lentiviral approach to induce long-term overexpression of IL-1ß in the dorsal hippocampus of adult male Sprague Dawley rats and examine its impact on cognition. Following three weeks of viral integration, pattern separation, a process involving hippocampal neurogenesis, was impaired in IL-1ß-treated rats in both object-location and touchscreen operant paradigms. This was coupled with a decrease in the number and neurite complexity of immature neurons in the hippocampus. Conversely, tasks involving the hippocampus, but not sensitive to disruption of hippocampal neurogenesis, including spontaneous alternation, novel object and location recognition were unaffected. Touchscreen operant visual discrimination, a cognitive task involving the prefrontal cortex, was largely unaffected by IL-1ß overexpression. In conclusion, these findings suggest that chronically elevated IL-1ß in the hippocampus selectively impairs pattern separation. Inflammatory-mediated disruption of adult hippocampal neurogenesis may contribute to the cognitive decline associated with neurodegenerative and stress-related disorders.

Social interaction-induced activation of RNA splicing in the amygdala of microbiome-deficient mice.

Social behaviour is regulated by activity of host-associated microbiota across multiple species. However, the molecular mechanisms mediating this relationship remain elusive. We therefore determined the dynamic, stimulus-dependent transcriptional regulation of germ-free (GF) and GF mice colonised post weaning (exGF) in the amygdala, a brain region critically involved in regulating social interaction. In GF mice the dynamic response seen in controls was attenuated and replaced by a marked increase in expression of splicing factors and alternative exon usage in GF mice upon stimulation, which was even more pronounced in exGF mice. In conclusion, we demonstrate a molecular basis for how the host microbiome is crucial for a normal behavioural response during social interaction. Our data further suggest that social behaviour is correlated with the gene-expression response in the amygdala, established during neurodevelopment as a result of host-microbe interactions. Our findings may help toward understanding neurodevelopmental events leading to social behaviour dysregulation, such as those found in autism spectrum disorders (ASDs).

The orphan nuclear receptor TLX regulates hippocampal transcriptome changes induced by IL-1ß.

TLX is an orphan nuclear receptor highly expressed within neural progenitor cells (NPCs) in the hippocampus where is regulates proliferation. Inflammation has been shown to have negative effects on hippocampal function as well as on NPC proliferation. Specifically, the pro-inflammatory cytokine IL-1ß suppresses NPC proliferation as well as TLX expression in the hippocampus. However, it is unknown whether TLX itself is involved in regulating the inflammatory response in the hippocampus. To explore the role of TLX in inflammation, we assessed changes in the transcriptional landscape of the hippocampus of TLX knockout mice (TLX-/-) compared to wildtype (WT) littermate controls with and without intrahippocampal injection of IL-1ß using a whole transcriptome RNA sequencing approach. We demonstrated that there is an increase in the transcription of genes involved in the promotion of inflammation and regulation of cell chemotaxis (Tnf, Il1b, Cxcr1, Cxcr2, Tlr4) and a decrease in the expression of genes relating to synaptic signalling (Lypd1, Syt4, Cplx2) in cannulated TLX-/- mice compared to WT controls. We demonstrate that mice lacking in TLX share a similar increase in 176 genes involved in regulating inflammation (e.g. Cxcl1, Tnf, Il1b) as WT mice injected with IL-1ß into the hippocampus. Moreover, TLX-/- mice injected with IL-1ß displayed a blunted transcriptional profile compared to WT mice injected with IL-1ß. Thus, TLX-/- mice, which already have an exaggerated inflammatory profile after cannulation surgery, are primed to respond differently to an inflammatory stimulus such as IL-1ß. Together, these results demonstrate that TLX regulates hippocampal inflammatory transcriptome response to brain injury (in this case cannulation surgery) and cytokine stimulation.

TLX is an intrinsic regulator of the negative effects of IL-1ß on proliferating hippocampal neural progenitor cells.

Hippocampal neurogenesis is a lifelong process whereby new neurons are produced and integrate into the host circuitry within the hippocampus. It is regulated by a multitude of extrinsic and intrinsic regulators and is believed to contribute to certain hippocampal-dependent cognitive tasks. Hippocampal neurogenesis and associated cognition have been demonstrated to be impaired after increases in the levels of proinflammatory cytokine IL-1ß in the hippocampus, such as that which occurs in various neurodegenerative and psychiatric disorders. IL-1ß also suppresses the expression of TLX (orphan nuclear receptor tailless homolog), which is an orphan nuclear receptor that functions to promote neural progenitor cell (NPC) proliferation and suppress neuronal differentiation; therefore, manipulation of TLX represents a potential strategy with which to prevent the antiproliferative effects of IL-1ß. In this study, we assessed the mechanism that underlies IL-1ß-induced changes in TLX expression and determined the protective capacity of TLX to mitigate the effects of IL-1ß on embryonic rat hippocampal neurosphere expansion. We demonstrate that IL-1ß activated the NF-κB pathway in proliferating NPCs and that this activation was responsible for IL-1ß-induced changes in TLX expression. In addition, we report that enhancing TLX expression prevented the IL-1ß-induced suppression of neurosphere expansion. Thus, we highlight TLX as a potential protective regulator of the antiproliferative effects of IL-1ß on hippocampal neurogenesis.-Ó'Léime, C. S., Kozareva, D. A., Hoban, A. E., Long-Smith, C. M., Cryan, J. F., Nolan, Y. M. TLX is an intrinsic regulator of the negative effects of IL-1ß on proliferating hippocampal neural progenitor cells.

Gutsy Moves: The Amygdala as a Critical Node in Microbiota to Brain Signaling.

The amygdala is a key brain area regulating responses to stress and emotional stimuli, so improving our understanding of how it is regulated could offer novel strategies for treating disturbances in emotion regulation. As we review here, a growing body of evidence indicates that the gut microbiota may contribute to a range of amygdala-dependent brain functions from pain sensitivity to social behavior, emotion regulation, and therefore, psychiatric health. In addition, it appears that the microbiota is necessary for normal development of the amygdala at both the structural and functional levels. While further investigations are needed to elucidate the exact mechanisms of microbiota-to-amygdala communication, ultimately, this work raises the intriguing possibility that the gut microbiota may become a viable treatment target in disorders associated with amygdala dysregulation, including visceral pain, post-traumatic stress disorder, and beyond. Also see the video abstract here: https://youtu.be/O5gvxVJjX18.

Microbial regulation of microRNA expression in the amygdala and prefrontal cortex.

BACKGROUND: There is growing evidence for a role of the gut microbiome in shaping behaviour relevant to many psychiatric and neurological disorders. Preclinical studies using germ-free (GF) animals have been essential in contributing to our current understanding of the potential importance of the host microbiome for neurodevelopment and behaviour. In particular, it has been repeatedly demonstrated that manipulation of the gut microbiome modulates anxiety-like behaviours. The neural circuits that underlie anxiety- and fear-related behaviours are complex and heavily depend on functional communication between the amygdala and prefrontal cortex (PFC). Previously, we have shown that the transcriptional networks within the amygdala and PFC of GF mice are altered. MicroRNAs (miRNAs) act through translational repression to control gene translation and have also been implicated in anxiety-like behaviours. However, it is unknown whether these features of host post-transcriptional machinery are also recruited by the gut microbiome to exert control over CNS transcriptional networks. RESULTS: We conducted Illumina® next-generation sequencing (NGS) in the amygdala and PFC of conventional, GF and germ-free colonized mice (exGF). We found a large proportion of miRNAs to be dysregulated in GF animals in both brain regions (103 in the amygdala and 31 in the PFC). Additionally, colonization of GF mice normalized some of the noted alterations. Next, we used a complementary approach to GF by manipulating the adult rat microbiome with an antibiotic cocktail to deplete the gut microbiota and found that this strategy also impacted the expression of relevant miRNAs. CONCLUSION: These results suggest that the microbiome is necessary for appropriate regulation of miRNA expression in brain regions implicated in anxiety-like behaviours.

Transferring the blues: Depression-associated gut microbiota induces neurobehavioural changes in the rat.

The gut microbiota interacts with the host via neuroimmune, neuroendocrine and neural pathways. These pathways are components of the brain-gut-microbiota axis and preclinical evidence suggests that the microbiota can recruit this bidirectional communication system to modulate brain development, function and behaviour. The pathophysiology of depression involves neuroimmune-neuroendocrine dysregulation. However, the extent to which changes in gut microbiota composition and function mediate the dysregulation of these pathways is unknown. Thirty four patients with major depression and 33 matched healthy controls were recruited. Cytokines, CRP, Salivary Cortisol and plasma Lipopolysaccharide binding protein were determined by ELISA. Plasma tryptophan and kynurenine were determined by HPLC. Fecal samples were collected for 16s rRNA sequencing. A Fecal Microbiota transplantation was prepared from a sub group of depressed patients and controls and transferred by oral gavage to a microbiota-deficient rat model. We demonstrate that depression is associated with decreased gut microbiota richness and diversity. Fecal microbiota transplantation from depressed patients to microbiota-depleted rats can induce behavioural and physiological features characteristic of depression in the recipient animals, including anhedonia and anxiety-like behaviours, as well as alterations in tryptophan metabolism. This suggests that the gut microbiota may play a causal role in the development of features of depression and may provide a tractable target in the treatment and prevention of this disorder.

Microbes & neurodevelopment--Absence of microbiota during early life increases activity-related transcriptional pathways in the amygdala.

The mammalian amygdala is a key emotional brain region for eliciting social behaviour, critically involved in anxiety and fear-related behaviours, and hence a focus of research on neurodevelopmental and stress-related disorders such as autism and anxiety. Recently, increasing evidence implicates host-microbe interactions in the aetiology of these conditions. Germ-free (GF) mice, devoid of any microbiota throughout organismal maturation, are a well-established tool to study the effects of absence of the microbiota on host physiology. A growing body of independently replicated findings confirm that GF animals demonstrate altered anxiety-related behaviour and impaired social behaviour. However, the underlying mechanisms of this interaction and the nature of the pathways involved are only insufficiently understood. To further elucidate the molecular underpinnings of microbe-brain interaction, we therefore exploited unbiased genome-wide transcriptional profiling to determine gene expression in the amygdala of GF and GF mice that have been colonised after weaning. Using RNA-sequencing and a comprehensive downstream analysis pipeline we studied the amygdala transcriptome and found significant differences at the levels of differential gene expression, exon usage and RNA-editing. Most surprisingly, we noticed upregulation of several immediate early response genes such as Fos, Fosb, Egr2 or Nr4a1 in association with increased CREB signalling in GF mice. In addition, we found differential expression and recoding of several genes implicated in brain physiology processes such as neurotransmission, neuronal plasticity, metabolism and morphology. In conclusion, our data suggest altered baseline neuronal activity in the amygdala of germ-free animals, which is established during early life and may have implications for understanding development and treatment of neurodevelopmental disorders.

Thinking small: towards microRNA-based therapeutics for anxiety disorders.

INTRODUCTION: Anxiety disorders are the most frequently diagnosed psychiatric conditions, negatively affecting quality of life and creating a significant economic burden. These complex disorders are extremely difficult to treat, and there is a great need for novel therapeutics with greater efficacy and minimal adverse side effects. AREAS COVERED: In this review, the authors describe the role that microribonucleic acids (microRNA or miRNA) play in the development of anxiety disorders and their potential to serve as biomarkers of disease as well as targets for pharmacological treatment. Furthermore, the authors discuss the current state of miRNA research, including both preclinical and clinical studies of anxiety disorders. EXPERT OPINION: There is mounting evidence that circulating miRNA may serve as biomarkers of disease and play a role in the development of disease, including psychiatric conditions such as anxiety disorders. Great strides have been made in cancer research, with miRNA-based therapies already in use in clinical studies. However, the use of miRNA for the treatment of neurological disorders, and psychiatric disorders in particular, is still in its nascent stage. The development of safe compounds that are able to cross the blood-brain barrier and target specific cell populations, which are relevant to anxiety-related neurocircuitry, is paramount for the emergence of novel, efficacious miRNA-based therapies in clinical settings.