Clostridioides difficile and neurological disorders: New perspectives.

Despite brain physiological functions or pathological dysfunctions relying on the activity of neuronal/non-neuronal populations, over the last decades a plethora of evidence unraveled the essential contribution of the microbial populations living and residing within the gut, called gut microbiota. The gut microbiota plays a role in brain (dys)functions, and it will become a promising valuable therapeutic target for several brain pathologies. In the present mini-review, after a brief overview of the role of gut microbiota in normal brain physiology and pathology, we focus on the role of the bacterium Clostridioides difficile, a pathogen responsible for recurrent and refractory infections, in people with neurological diseases, summarizing recent correlative and causative evidence in the scientific literature and highlighting the potential of microbiota-based strategies targeting this pathogen to ameliorate not only gastrointestinal but also the neurological symptoms.

Fecal Microbiota Transplantation as New Therapeutic Avenue for Human Diseases.

The human body is home to a variety of micro-organisms. Most of these microbial communities reside in the gut and are referred to as gut microbiota. Over the last decades, compelling evidence showed that a number of human pathologies are associated with microbiota dysbiosis, thereby suggesting that the reinstatement of physiological microflora balance and composition might ameliorate the clinical symptoms. Among possible microbiota-targeted interventions, pre/pro-biotics supplementations were shown to provide effective results, but the main limitation remains in the limited microbial species available as probiotics. Differently, fecal microbiota transplantation involves the transplantation of a solution of fecal matter from a donor into the intestinal tract of a recipient in order to directly change the recipient's gut microbial composition aiming to confer a health benefit. Firstly used in the 4th century in traditional Chinese medicine, nowadays, it has been exploited so far to treat recurrent Clostridioides difficile infections, but accumulating data coming from a number of clinical trials clearly indicate that fecal microbiota transplantation may also carry the therapeutic potential for a number of other conditions ranging from gastrointestinal to liver diseases, from cancer to inflammatory, infectious, autoimmune diseases and brain disorders, obesity, and metabolic syndrome. In this review, we will summarize the commonly used preparation and delivery methods, comprehensively review the evidence obtained in clinical trials in different human conditions and discuss the variability in the results and the pivotal importance of donor selection. The final aim is to stimulate discussion and open new therapeutic perspectives among experts in the use of fecal microbiota transplantation not only in Clostridioides difficile infection but as one of the first strategies to be used to ameliorate a number of human conditions.

Gut and Brain: Investigating Physiological and Pathological Interactions Between Microbiota and Brain to Gain New Therapeutic Avenues for Brain Diseases.

Brain physiological functions or pathological dysfunctions do surely depend on the activity of both neuronal and non-neuronal populations. Nevertheless, over the last decades, compelling and fast accumulating evidence showed that the brain is not alone. Indeed, the so-called "gut brain," composed of the microbial populations living in the gut, forms a symbiotic superorganism weighing as the human brain and strongly communicating with the latter via the gut-brain axis. The gut brain does exert a control on brain (dys)functions and it will eventually become a promising valuable therapeutic target for a number of brain pathologies. In the present review, we will first describe the role of gut microbiota in normal brain physiology from neurodevelopment till adulthood, and thereafter we will discuss evidence from the literature showing how gut microbiota alterations are a signature in a number of brain pathologies ranging from neurodevelopmental to neurodegenerative disorders, and how pre/probiotic supplement interventions aimed to correct the altered dysbiosis in pathological conditions may represent a valuable future therapeutic strategy.

5-HT/GABA interaction in epilepsy.

Epilepsy is a neurological condition characterized by synchronous neuronal oscillations (seizures) in the electroencephalogram. Seizures are classified in focal or generalized (depending on the brain territory interested during seizures), and in convulsive and/or not convulsive (depending on the presence or not of involuntary movements). The current pharmacological treatments are mainly based on GABA modulation although different neurotransmitters are also involved in epilepsy, including serotonin. However despite much extensive progress in the understanding of epilepsy mechanisms, still, a percentage of people with epilepsy are pharmaco-resistant calling for the need for new therapeutic targets. Here we review preclinical and human evidence showing that serotonin modulates epilepsy that this likely happens via a major modulation/interaction with GABA.

5-HT/GABA interaction in neurodevelopment and plasticity.

The adult brain is the result of a multistages complex neurodevelopmental process involving genetic, molecular and microenvironmental factors as well as diverse patterns of electrical activity. In the postnatal life, immature neuronal circuits undergo an experience-dependent maturation during critical periods of plasticity, but the brain still retains plasticity during adult life. In all these stages, the neurotransmitter GABA plays a pivotal role. In this chapter, we will describe the interaction of 5-HT with GABA in regulating neurodevelopment and plasticity.

The impact of chronic daily nicotine exposure and its overnight withdrawal on the structure of anxiety-related behaviors in rats: Role of the lateral habenula.

Tobacco smoking is a serious health problem worldwide and a leading cause of mortality. Nicotine, the addictive component of tobacco, affects a range of emotional responses, including anxiety-related behaviors. Although perceived by smokers to be anxiolytic, evidence suggests that smoking increases anxiety and that mood fluctuates with nicotine intake. Thus, nicotine addiction may depend on easing the psychobiological distress caused by its abuse. The lateral habenula (LHb) has been implicated as a neural substrate for acute nicotine-induced anxiety, but its role in anxiety-like behaviors associated with chronic nicotine exposure has not been explored. Here, we assessed the effect of chronic nicotine exposure and its subsequent overnight withdrawal on anxiety-like behavior using both quantitative and multivariate T-pattern analysis in rats tested using the hole-board apparatus. Additionally, we explored the role of the LHb by comparing the behavioral effects of short-term nicotine withdrawal in chronically treated LHb-lesioned rats. Quantitative analysis revealed increased anxiety-like behavior in chronically treated overnight nicotine-deprived rats, as manifested in reduced general and focused exploratory behaviors, which was eased in animals that received nicotine. Quantitative analysis failed to reveal a role of the LHb in overnight nicotine deprivation-induced anxiety. Conversely, T-pattern analysis of behavioral outcomes revealed that chronic nicotine-treated rats still show anxiety-like behavior following nicotine challenge. Moreover, it demonstrated that the LHb lesion induced a stronger anxiolytic-like response to the acute challenge of nicotine in chronically nicotine-exposed animals, implicating the LHb in the anxiogenic effect of chronic nicotine exposure. These data further highlight the LHb as a promising target for smoking cessation therapies and support the importance of T-pattern analysis for behavioral analysis.

Acute and Chronic Nicotine Exposures Differentially Affect Central Serotonin 2A Receptor Function: Focus on the Lateral Habenula.

Nicotine addiction is a serious public health problem causing millions of deaths worldwide. Serotonin (5-hydroxytryptamine; 5-HT) is involved in central nervous system (CNS) nicotine effects, and it has been suggested as a promising pharmacological target for smoking cessation. In this regard, what is particularly interesting are the 5-HT2A receptors (5-HT2ARs) and the lateral habenula (LHb), a central area in nicotine addiction that we showed to be under a strong 5-HT2AR-modulation. Single-cell extracellular recording of LHb neurons was used to study the 5-HT2AR function by intravenously administrating the potent agonist TCB-2. Acute nicotine (2 mg/kg, intraperitoneal, i.p.) and chronic nicotine (6 mg/kg/day for 14 days) differently affected both the 5-HT2AR-immuno reactive (IR) neuron number and the 5-HT2AR immunostaining area in the different brain areas studied. After acute nicotine, TCB-2 cumulative doses (5-640 µg/kg, intravenous, i.v.) bidirectionally affected the activity of 74% of LHb recorded neurons. After chronic nicotine treatment, TCB-2 was only capable of decreasing the LHb firing rate. The expression of 5-HT2AR under acute and chronic nicotine exposure was studied in the LHb and in other brain areas involved in nicotine effects in rats by using immunohistochemistry. These data reveal that acute and chronic nicotine differentially affect the 5-HT2AR function in different brain areas and this might be relevant in nicotine addiction and its treatment.

Effects of chronic nicotine on the temporal structure of anxiety-related behavior in rats tested in hole-board.

The present study aimed to assess the behavioral effects of chronic treatments of different doses of nicotine by using both quantitative and multivariate T-pattern analysis (TPA), which can reveal hidden behavioral structures, in Sprague-Dawley rats tested in the hole-board apparatus. To this purpose, nicotine ditartrate was administered at the doses of 0.1, 0.5 and 1 mg/kg i.p., three times per day, for 14 consecutive days. As to quantitative evaluations, we observed significant reductions in the mean durations and mean frequencies of walking, climbing, immobile-sniffing and rearing in comparison to control. A significant reduction of edge-sniff and head-dip mean frequencies was also detected for all the doses tested. TPA revealed an increase in the number and the mean length of different T-patterns induced by the three doses of nicotine. On the other hand, a significant reduction of the mean occurrences of T-patterns was revealed. Overall, our results obtained by using both quantitative and T-pattern analyses indicate that chronic nicotine induces an anxiety condition characterized by a behavioral re-organization orbiting around the two main components of hole exploration, that is, head-dip and edge-sniff. A better understanding of the link between nicotine and anxiety might help to find new therapies for smoking cessation.

Lorcaserin bidirectionally regulates dopaminergic function site-dependently and disrupts dopamine brain area correlations in rats.

Lorcaserin, which is a selective agonist of serotonin2C receptors (5-HT2CRs), is a new FDA-approved anti-obesity drug that has also shown therapeutic promise in other brain disorders, such as addiction and epilepsy. The modulation of dopaminergic function might be critical in the therapeutic effect of lorcaserin, but its exact effect is unknown. Here, we studied the effect of the peripheral administration of lorcaserin on the ventral tegmental area (VTA), the substantia nigra pars compacta (SNc) dopaminergic neural activity, dopamine (DA) dialysis levels in the nucleus accumbens and striatum and on DA tissue levels in 29 different rat brain regions. Lorcaserin (5-640 µg/kg, i.v.) moderately inhibited only a subpopulation of VTA DA neurons, but had no effect on the SNc neurons. Lorcaserin (0.3, 3 mg/kg, i.p.) did not change VTA and SNc DA population neural activity but slightly decreased the firing rate and burst firing of the spontaneously active VTA neurons, without altering DA extracellular dialysate levels in both the nucleus accumbens and the striatum. Quantitative analysis of DA and metabolites tissue contents of the 29 areas studied revealed that lorcaserin (0.3 or 3 mg/kg, i.p.) only affected a few brain regions, i.e., increased DA in the central amygdala, ventral hypothalamus and nucleus accumbens core and decreased it in the ventromedial striatum. On the other hand, lorcaserin dramatically changed the direction and reduced the number of correlations of DA tissue content among several brain areas. These effects on DA terminal networks might be significant in the therapeutic mechanism of lorcaserin. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Synergistic action of CB1 and 5-HT2B receptors in preventing pilocarpine-induced status epilepticus in rats.

Endocannabinoids (eCBs) and serotonin (5-HT) play a neuromodulatory role in the central nervous system. Both eCBs and 5-HT regulate neuronal excitability and their pharmacological potentiation has been shown to control seizures in pre-clinical and human studies. Compelling evidence indicates that eCB and 5-HT systems interact to modulate several physiological and pathological brain functions, such as food intake, pain, drug addiction, depression, and anxiety. Nevertheless, there is no evidence of an eCB/5-HT interaction in experimental and human epilepsies, including status epilepticus (SE). Here, we performed video-EEG recording in behaving rats treated with the pro-convulsant agent pilocarpine (PILO), in order to study the effect of the activation of CB1/5-HT2 receptors and their interaction on SE. Synthetic cannabinoid agonist WIN55,212-2 (WIN) decreased behavioral seizure severity of PILO-induced SE at 2 mg/kg (but not at 1 and 5 mg/kg, i.p.), while 5-HT2B/2C receptor agonist RO60-0175 (RO; 1, 3, 10 mg/kg, i.p.) was devoid of any effect. RO 3 mg/kg was instead capable of potentiating the effect of WIN 2 mg/kg on the Racine scale score. Surprisingly, neither WIN 2 mg/kg nor RO 3 mg/kg had any effect on the incidence and the intensity of EEG seizures when administered alone. However, WIN+RO co-administration reduced the incidence and the severity of EEG SE and increased the latency to SE onset after PILO injection. WIN+RO effects were blocked by the selective CB1R antagonist AM251 and the 5-HT2BR antagonist RS127445, but not by the 5-HT2CR antagonist SB242084 or the 5-HT2AR antagonist MDL11,939. These data revealed a synergistic interaction between CB1R/5-HT2BR in the expression of PILO-induced SE.

Aerobic exercise and a BDNF-mimetic therapy rescue learning and memory in a mouse model of Down syndrome.

Down syndrome (DS) is caused by the triplication of human chromosome 21 and represents the most frequent genetic cause of intellectual disability. The trisomic Ts65Dn mouse model of DS shows synaptic deficits and reproduces the essential cognitive disabilities of the human syndrome. Aerobic exercise improved various neurophysiological dysfunctions in Ts65Dn mice, including hippocampal synaptic deficits, by promoting synaptogenesis and neurotransmission at glutamatergic terminals. Most importantly, the same intervention also prompted the recovery of hippocampal adult neurogenesis and synaptic plasticity and restored cognitive performance in trisomic mice. Additionally, the expression of brain-derived neurotrophic factor (BDNF) was markedly decreased in the hippocampus of patients with DS. Since the positive effect of exercise was paralleled by increased BDNF expression in trisomic mice, we investigated the effectiveness of a BDNF-mimetic treatment with 7,8-dihydroxyflavone at alleviating intellectual disabilities in the DS model. Pharmacological stimulation of BDNF signaling rescued synaptic plasticity and memory deficits in Ts65Dn mice. Based on our findings, Ts65Dn mice benefit from interventions aimed at promoting brain plasticity, and we provide evidence that BDNF signaling represents a potentially new pharmacological target for treatments aimed at rescuing cognitive disabilities in patients with DS.

Early IGF-1 primes visual cortex maturation and accelerates developmental switch between NKCC1 and KCC2 chloride transporters in enriched animals.

Environmental enrichment (EE) has a remarkable impact on brain development. Continuous exposure to EE from birth determines a significant acceleration of visual system maturation both at retinal and cortical levels. A pre-weaning enriched experience is sufficient to trigger the accelerated maturation of the visual system, suggesting that factors affected by EE during the first days of life might prime visual circuits towards a faster development. The search for such factors is crucial not only to gain a better understanding of the molecular hierarchy of brain development but also to identify molecular pathways amenable to be targeted to correct atypical brain developmental trajectories. Here, we showed that IGF-1 levels are increased in the visual cortex of EE rats as early as P6 and this is a crucial event for setting in motion the developmental program induced by EE. Early intracerebroventricular (i.c.v.) infusion of IGF-1 in standard rats was sufficient to mimic the action of EE on visual acuity development, whereas blocking IGF-1 signaling by i.c.v. injections of the IGF-1 receptor antagonist JB1 prevented the deployment of EE effects. Early IGF-1 decreased the ratio between the expression of NKCC1 and KCC2 cation/chloride transporters, and the reversal potential for GABAAR-driven Cl- currents (ECl) was shifted toward more negative potentials, indicating that IGF-1 is a crucial factor in accelerating the maturation of GABAergic neurotransmission and promoting the developmental switch of GABA polarity from excitation to inhibition. In addition, early IGF-1 promoted a later occurring increase in its own expression, suggesting a priming effect of early IGF-1 in driving post-weaning cortical maturation.

Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability, and altered GABAergic transmission through Cl(-)-permeable GABAA receptors (GABAARs) contributes considerably to learning and memory deficits in DS mouse models. However, the efficacy of GABAergic transmission has never been directly assessed in DS. Here GABAAR signaling was found to be excitatory rather than inhibitory, and the reversal potential for GABAAR-driven Cl(-) currents (ECl) was shifted toward more positive potentials in the hippocampi of adult DS mice. Accordingly, hippocampal expression of the cation Cl(-) cotransporter NKCC1 was increased in both trisomic mice and individuals with DS. Notably, NKCC1 inhibition by the FDA-approved drug bumetanide restored ECl, synaptic plasticity and hippocampus-dependent memory in adult DS mice. Our findings demonstrate that GABA is excitatory in adult DS mice and identify a new therapeutic approach for the potential rescue of cognitive disabilities in individuals with DS.

Early depolarizing GABA controls critical-period plasticity in the rat visual cortex.

Hyperpolarizing and inhibitory GABA regulates critical periods for plasticity in sensory cortices. Here we examine the role of early, depolarizing GABA in the control of plasticity mechanisms. We report that brief interference with depolarizing GABA during early development prolonged critical-period plasticity in visual cortical circuits without affecting the overall development of the visual system. The effects on plasticity were accompanied by dampened inhibitory neurotransmission, downregulation of brain-derived neurotrophic factor (BDNF) expression and reduced density of extracellular matrix perineuronal nets. Early interference with depolarizing GABA decreased perinatal BDNF signaling, and a pharmacological increase of BDNF signaling during GABA interference rescued the effects on plasticity and its regulators later in life. We conclude that depolarizing GABA exerts a long-lasting, selective modulation of plasticity of cortical circuits by a strong crosstalk with BDNF.

Modulation of GABAergic transmission in development and neurodevelopmental disorders: investigating physiology and pathology to gain therapeutic perspectives.

During mammalian ontogenesis, the neurotransmitter GABA is a fundamental regulator of neuronal networks. In neuronal development, GABAergic signaling regulates neural proliferation, migration, differentiation, and neuronal-network wiring. In the adult, GABA orchestrates the activity of different neuronal cell-types largely interconnected, by powerfully modulating synaptic activity. GABA exerts these functions by binding to chloride-permeable ionotropic GABAA receptors and metabotropic GABAB receptors. According to its functional importance during development, GABA is implicated in a number of neurodevelopmental disorders such as autism, Fragile X, Rett syndrome, Down syndrome, schizophrenia, Tourette's syndrome and neurofibromatosis. The strength and polarity of GABAergic transmission is continuously modulated during physiological, but also pathological conditions. For GABAergic transmission through GABAA receptors, strength regulation is achieved by different mechanisms such as modulation of GABAA receptors themselves, variation of intracellular chloride concentration, and alteration in GABA metabolism. In the never-ending effort to find possible treatments for GABA-related neurological diseases, of great importance would be modulating GABAergic transmission in a safe and possibly physiological way, without the dangers of either silencing network activity or causing epileptic seizures. In this review, we will discuss the different ways to modulate GABAergic transmission normally at work both during physiological and pathological conditions. Our aim is to highlight new research perspectives for therapeutic treatments that reinstate natural and physiological brain functions in neuro-pathological conditions.

High-performance and site-directed in utero electroporation by a triple-electrode probe.

In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here we present a new in utero electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum.