Patrick Fitzgerald

The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner

Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous system (CNS) signalling. Regulation of the microbiome–gut–brain axis is essential for maintaining homeostasis, including that of the CNS. However, there is a paucity of data pertaining to the influence of microbiome on the serotonergic system. Germ-free (GF) animals represent an effective preclinical tool to investigate such phenomena. Here we show that male GF animals have a significant elevation in the hippocampal concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, its main metabolite, compared with conventionally colonised control animals. Moreover, this alteration is sex specific in contrast with the immunological and neuroendocrine effects which are evident in both sexes. Concentrations of tryptophan, the precursor of serotonin, are increased in the plasma of male GF animals, suggesting a humoral route through which the microbiota can influence CNS serotonergic neurotransmission. Interestingly, colonisation of the GF animals post weaning is insufficient to reverse the CNS neurochemical consequences in adulthood of an absent microbiota in early life despite the peripheral availability of tryptophan being restored to baseline values. In addition, reduced anxiety in GF animals is also normalised following restoration of the intestinal microbiota. These results demonstrate that CNS neurotransmission can be profoundly disturbed by the absence of a normal gut microbiota and that this aberrant neurochemical, but not behavioural, profile is resistant to restoration of a normal gut flora in later life.

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.

The probiotic Bifidobacterium infantis 35624 displays visceral antinociceptive effects in the rat

Background  Irritable bowel syndrome (IBS) is characterized by recurrent abdominal pain and altering bowel habit with a high percentage of patients displaying comorbid anxiety. Growing clinical and preclinical evidence suggests that probiotic agents may restore the altered brain–gut communication in IBS. In this study, we evaluated the efficacy of repeated treatment with three different probiotics in reducing visceral pain in visceral normosensitive (Sprague–Dawley [SD]) and visceral hypersensitive (Wistar–Kyoto [WKY]) rat strains.

Tryptophan catabolism in females with irritable bowel syndrome: relationship to interferon‐gamma, severity of symptoms and psychiatric co‐morbidity

Abstract  Irritable bowel syndrome (IBS) has been linked with abnormal serotonin functioning and immune activation. Tryptophan forms the substrate for serotonin biosynthesis, but it can alternatively be catabolized to kynurenine (Kyn) by the enzyme indoleamine 2,3‐dioxygenase (IDO), the main inducer of which is interferon‐gamma. The primary aim of this study was to test the hypothesis that IBS is associated with increased tryptophan (Trp) catabolism along the Kyn pathway due to increased IFN‐γ levels. Plasma Kyn, Trp and IFN‐γ levels were measured in 41 female IBS subjects and 33 controls. Indoleamine 2,3‐dioxygenase activity was assessed using the Kyn to Trp ratio. Psychiatric co‐morbidity was assessed using the Patient Health Questionnaire, and severity of IBS assessed using self‐report ordinal scales. Irritable bowel syndrome subjects had increased Kyn concentrations compared with controls (P = 0.039) and there was a trend for Kyn:Trp to be increased in the IBS group (P = 0.09). There was a positive correlation between IBS severity and Kyn:Trp (r = 0.57, P < 0.001). Those with severe IBS symptoms had increased Kyn:Trp (P < 0.005) compared to those with less severe symptoms and controls, and were over twice as likely to have depression or anxiety compared to those with less severe IBS (RR = 2.2, 95% CI 1.2–3.9). No difference in IFN‐γ levels was observed between groups; however, IFN‐γ was positively correlated with Kyn:Trp in IBS (r = 0.58, P = 0.005) but not controls (r = 0.12, P = 0.5). Females with IBS have abnormal Trp catabolism. The Kyn:Trp is related to symptom severity, and those with severe IBS symptoms have increased shunting of Trp along the Kyn pathway which contributes to the abnormal serotonergic functioning in this syndrome.

5-HT(2B) receptors modulate visceral hypersensitivity in a stress-sensitive animal model of brain-gut axis dysfunction.

Background  Irritable bowel syndrome (IBS) is associated with an enhanced perception to visceral stimuli and exaggerated stress response. The serotonergic neurotransmitter system has been strongly implicated as a key player in the manifestation of IBS symptomatology including visceral hypersensitivity. However the role of 5‐HT2B receptors in visceral pain, although speculated, is currently unclear. Thus we assessed the impact of a selective 5‐HT2B receptor antagonist, RS‐127445, on visceral hypersensitivity in a model of brain gut axis dysfunction the Wistar Kyoto (WKY) rat.

Neuroprotective effects of novel phosphatidylglycerol-based phospholipids in the 6-hydroxydopamine model of Parkinson's disease

Administration of VP025 (Vasogen Inc.), a novel drug formulation based on phospholipid nanoparticles incorporating phosphatidylglycerol, has previously been shown to have a neuroprotective effect in the brain. We examined the effect of VP025 in a rat model of Parkinsons disease, the 6‐hydroxydopamine (6‐OHDA) lesion of the medial forebrain bundle. VP025 or phosphate‐buffered saline (PBS) was administered to rats 14 days, 13 days and 1 day before the unilateral 6‐OHDA lesion. Functional integrity of nigrostriatal dopaminergic neurons was assessed 7 and 21 days later by amphetamine‐induced rotational testing and we observed that rotational counts were significantly less in rats that were pretreated with VP025 compared with PBS‐pretreated 6‐OHDA‐lesioned rats. Neurochemical analysis at 10 and 28 days after lesion revealed that VP025 protected against a 6‐OHDA‐induced decrease in concentrations of striatal dopamine and its metabolites. Immunocytochemical studies of the ipsilateral substantia nigra showed that VP025 significantly inhibited 6‐OHDA‐induced loss of dopaminergic neurons. We also observed that increases in immunostaining for activated microglia and for activated p38 in dopaminergic neurons of 6‐OHDA‐lesioned rats were prevented by VP025. This study shows that VP025 has significant protective effects on the 6‐OHDA‐lesioned nigrostriatal pathway and may therefore have potential for the treatment of Parkinsons disease.

BDNF expression in the hippocampus of maternally separated rats: does Bifidobacterium breve 6330 alter BDNF levels?

Brain-derived neurotrophic factor (BDNF) is of interest because of its putative role in stress and psychiatric disorders. Maternal separation is used as an animal model of early-life stress and of irritable bowel syndrome (IBS). Animals exposed to the paradigm show altered gut function together with heightened levels of arousal and corticosterone. Some probiotic organisms have been shown to be of benefit in IBS and influence the brain-gut axis. Our objective was to investigate the effects of maternal separation on BDNF under basal conditions and in response to the probiotic Bifidobacterium breve 6330. The study implemented the maternal separation model which we have previously described. Polymerase chain reaction and in situ hybridisation were performed to measure the effect of maternal separation on both BDNF total variants and BDNF splice variant (exon) IV in the hippocampus. Maternally separated and non-separated rats were treated with B. breve 6330, to investigate the effect of this probiotic on BDNF total variant and BDNF exon IV expression. Maternal separation increased BDNF total variants (P<0.01), whilst having no effect on BDNF exon IV. B. breve 6330 increased BDNF total variants (P<0.01), and decreased BDNF splice variant IV, in non-separated rats (P<0.01). B. breve 6330 did not alter BDNF levels in the maternally separated rats. Maternal separation caused a marked increase in BDNF in the hippocampus. While B. breve 6330 influenced BDNF in normal animals, it had no significant effect on BDNF in those which were maternally separated. We have demonstrated that an orally administered probiotic can influence hippocampal BDNF.

Inhibition of P-glycoprotein enhances transport of imipramine across the blood–brain barrier: microdialysis studies in conscious freely moving rats

Recent studies indicate that efflux of antidepressants by the multidrug resistance transporter P‐glycoprotein (P‐gp) at the blood–brain barrier (BBB) may contribute to treatment‐resistant depression (TRD) by limiting intracerebral antidepressant concentrations. In addition, clinical experience shows that adjunctive treatment with the P‐gp inhibitor verapamil may improve the clinical outcome in TRD. Therefore, the present study aimed to investigate the effect of P‐gp inhibition on the transport of the tricyclic antidepressant imipramine and its active metabolite desipramine across the BBB.

Rodent Models of Colorectal Distension

Colorectal distension (CRD) is a widely accepted, reproducible method for assessing visceral sensitivity in both clinical and pre‐clinical studies. Distension of the colon mirrors the human scenario of visceral pain with regard to intensity and referral of pain in patients. There are several readouts that can be applied to the CRD protocol depending on the species being evaluated, two of which are described in this unit. CRD can be used to measure the impact of novel compounds, strain, or genetic differences as well as the effect of physical and psychological stressors on the sensitivity of the colon. Investigation of the impact of a noxious visceral stimulus (CRD) on other systems within the body can also be carried out. Given that visceral pain is a major clinical problem and one of the most common reasons patients seek out medical advice, the ability to assess this type of pain is essential to the discovery of successful treatments. This unit outlines two protocols that describe CRD of rats and mice. Curr. Protoc. Neurosci. 61:9.40.1‐9.40.13.

Bifidobacterium breve with α-Linolenic Acid and Linoleic Acid Alters Fatty Acid Metabolism in the Maternal Separation Model of Irritable Bowel Syndrome

The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (109 microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.