From the Microbiome to the Electrome: Implications for the Microbiota-Gut-Brain Axis.

The gut microbiome plays a fundamental role in metabolism, as well as the immune and nervous systems. Microbial imbalance (dysbiosis) can contribute to subsequent physical and mental pathologies. As such, interest has been growing in the microbiota-gut-brain brain axis and the bioelectrical communication that could exist between bacterial and nervous cells. The aim of this study was to investigate the bioelectrical profile (electrome) of two bacterial species characteristic of the gut microbiome: a Proteobacteria Gram-negative bacillus Escherichia coli (E. coli), and a Firmicutes Gram-positive coccus Enterococcus faecalis (E. faecalis). We analyzed both bacterial strains to (i) validate the fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), as a reliable reporter of the changes in membrane potential (Vmem) for both bacteria; (ii) assess the evolution of the bioelectric profile throughout the growth of both strains; (iii) investigate the effects of two neural-type stimuli on Vmem changes: the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA); (iv) examine the impact of the bioelectrical changes induced by neurotransmitters on bacterial growth, viability, and cultivability using absorbance, live/dead fluorescent probes, and viable counts, respectively. Our findings reveal distinct bioelectrical profiles characteristic of each bacterial species and growth phase. Importantly, neural-type stimuli induce Vmem changes without affecting bacterial growth, viability, or cultivability, suggesting a specific bioelectrical response in bacterial cells to neurotransmitter cues. These results contribute to understanding the bacterial response to external stimuli, with potential implications for modulating bacterial bioelectricity as a novel therapeutic target.

Bioelectrical State of Bacteria Is Linked to Growth Dynamics and Response to Neurotransmitters: Perspectives for the Investigation of the Microbiota-Brain Axis.

Inter-cellular communication is mediated by a sum of biochemical, biophysical, and bioelectrical signals. This might occur not only between cells belonging to the same tissue and/or animal species but also between cells that are, from an evolutionary point of view, far away. The possibility that bioelectrical communication takes place between bacteria and nerve cells has opened exciting perspectives in the study of the gut microbiota-brain axis. The aim of this paper is (i) to establish a reliable method for the assessment of the bioelectrical state of two bacterial strains: Bacillus subtilis (B. subtilis) and Limosilactobacillus reuteri (L. reuteri); (ii) to monitor the bacterial bioelectrical profile throughout its growth dynamics; and (iii) to evaluate the effects of two neurotransmitters (glutamate and γ-aminobutyric acid-GABA) on the bioelectrical signature of bacteria. Our results show that membrane potential (Vmem) and the proliferative capacity of the population are functionally linked in B. subtilis in each phase of the cell cycle. Remarkably, we demonstrate that bacteria respond to neural signals by changing Vmem properties. Finally, we show that Vmem changes in response to neural stimuli are present also in a microbiota-related strain L. reuteri. Our proof-of-principle data reveal a new methodological approach for the better understanding of the relation between bacteria and the brain, with a special focus on gut microbiota. Likewise, this approach will open exciting perspectives in the study of the inter-cellular mechanisms which regulate the bi-directional communication between bacteria and neurons and, ultimately, for designing gut microbiota-brain axis-targeted treatments for neuropsychiatric diseases.

Gut Microbiota and Neuroplasticity.

The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota-gut-brain axis) has led to a paradigm shift in the neurosciences. Understanding the neurobiological mechanisms supporting the relevance of actions mediated by the gut microbiota for brain physiology and neuronal functioning is a key research area. In this review, we discuss the literature showing how the microbiota is emerging as a key regulator of the brain's function and behavior, as increasing amounts of evidence on the importance of the bidirectional communication between the intestinal bacteria and the brain have accumulated. Based on recent discoveries, we suggest that the interaction between diet and the gut microbiota, which might ultimately affect the brain, represents an unprecedented stimulus for conducting new research that links food and mood. We also review the limited work in the clinical arena to date, and we propose novel approaches for deciphering the gut microbiota-brain axis and, eventually, for manipulating this relationship to boost mental wellness.

An in vivo brain-bacteria interface: the developing brain as a key regulator of innate immunity.

Infections have numerous effects on the brain. However, possible roles of the brain in protecting against infection, and the developmental origin and role of brain signaling in immune response, are largely unknown. We exploited a unique Xenopus embryonic model to reveal control of innate immune response to pathogenic E. coli by the developing brain. Using survival assays, morphological analysis of innate immune cells and apoptosis, and RNA-seq, we analyzed combinations of infection, brain removal, and tail-regenerative response. Without a brain, survival of embryos injected with bacteria decreased significantly. The protective effect of the developing brain was mediated by decrease of the infection-induced damage and of apoptosis, and increase of macrophage migration, as well as suppression of the transcriptional consequences of the infection, all of which decrease susceptibility to pathogen. Functional and pharmacological assays implicated dopamine signaling in the bacteria-brain-immune crosstalk. Our data establish a model that reveals the very early brain to be a central player in innate immunity, identify the developmental origins of brain-immune interactions, and suggest several targets for immune therapies.

Dependence of Neuroprosthetic Stimulation on the Sensory Modality of the Trigeminal Neurons Following Nerve Injury. Implications in the Design of Future Sensory Neuroprostheses for Correct Perception and Modulation of Neuropathic Pain.

Amputation of a sensory peripheral nerve induces severe anatomical and functional changes along the afferent pathway as well as perception alterations and neuropathic pain. In previous studies we showed that electrical stimulation applied to a transected infraorbital nerve protects the somatosensory cortex from the above-mentioned sensory deprivation-related changes. In the present study we focus on the initial tract of the somatosensory pathway and we investigate the way weak electrical stimulation modulates the neuroprotective-neuroregenerative and functional processes of trigeminal ganglia primary sensory neurons by studying the expression of neurotrophins (NTFs) and Glia-Derived Neurotrophic Factors (GDNFs) receptors. Neurostimulation was applied to the proximal stump of a transected left infraorbitary nerve using a neuroprosthetic micro-device 12 h/day for 4 weeks in freely behaving rats. Neurons were studied by in situ hybridization and immunohistochemistry against RET (proto-oncogene tyrosine kinase "rearranged during transfection"), tropomyosin-related kinases (TrkA, TrkB, TrkC) receptors and IB4 (Isolectin B4 from Griffonia simplicifolia). Intra-group (left vs. right ganglia) and inter-group comparisons (between Control, Axotomization and Stimulation-after-axotomization groups) were performed using the mean percentage change of the number of positive cells per section [100∗(left-right)/right)]. Intra-group differences were studied by paired t-tests. For inter-group comparisons ANOVA test followed by post hoc LSD test (when P < 0.05) were used. Significance level (α) was set to 0.05 in all cases. Results showed that (i) neurostimulation has heterogeneous effects on primary nociceptive and mechanoceptive/proprioceptive neurons; (ii) neurostimulation affects RET-expressing small and large neurons which include thermo-nociceptors and mechanoceptors, as well as on the IB4- and TrkB-positive populations, which mainly correspond to non-peptidergic thermo-nociceptive cells and mechanoceptors respectively. Our results suggest (i) electrical stimulation differentially affects modality-specific primary sensory neurons (ii) artificial input mainly acts on specific nociceptive and mechanoceptive neurons (iii) neuroprosthetic stimulation could be used to modulate peripheral nerve injuries-induced neuropathic pain. These could have important functional implications in both, the design of effective clinical neurostimulation-based protocols and the development of neuroprosthetic devices, controlling primary sensory neurons through selective neurostimulation.

Brief Local Application of Progesterone via a Wearable Bioreactor Induces Long-Term Regenerative Response in Adult Xenopus Hindlimb.

The induction of limb repair in adult vertebrates is a pressing, unsolved problem. Here, we characterize the effects of an integrated device that delivers drugs to severed hindlimbs of adult Xenopus laevis, which normally regenerate cartilaginous spikes after amputation. A wearable bioreactor containing a silk protein-based hydrogel that delivered progesterone to the wound site immediately after hindlimb amputation for only 24 hr induced the regeneration of paddle-like structures in adult frogs. Molecular markers, morphometric analysis, X-ray imaging, immunofluorescence, and behavioral assays were used to characterize the differences between the paddle-like structures of successful regenerates and hypomorphic spikes that grew in untreated animals. Our experiments establish a model for testing therapeutic cocktails in vertebrate hindlimb regeneration, identify pro-regenerative activities of progesterone-containing bioreactors, and provide proof of principle of brief use of integrated device-based delivery of small-molecule drugs as a viable strategy to induce and maintain a long-term regenerative response.

Booting up the organism during development: Pre-behavioral functions of the vertebrate brain in guiding body morphogenesis.

A recent study in Xenopus laevis embryos showed that the very early brain has important functions long before behavior. While the nascent brain is being constructed, it is required for normal patterning of the muscle and peripheral nerve networks, including those far away from the head. In addition to providing important developmental signals to remote tissues in normal embryogenesis, its presence is also able to render harmless exposure to specific chemicals that normally act as teratogens. These activities of the early brain can be partially compensated for in a brainless embryo by experimental modulation of neurotransmitter and ion channel signaling. Here, we discuss the major findings of this paper in the broader context of developmental physiology, neuroscience, and biomedicine. This novel function of the embryonic brain has significant implications, especially for understanding developmental toxicology and teratogenesis in the context of pharmaceutical and environmental reagents.

The brain is required for normal muscle and nerve patterning during early Xenopus development.

Possible roles of brain-derived signals in the regulation of embryogenesis are unknown. Here we use an amputation assay in Xenopus laevis to show that absence of brain alters subsequent muscle and peripheral nerve patterning during early development. The muscle phenotype can be rescued by an antagonist of muscarinic acetylcholine receptors. The observed defects occur at considerable distances from the head, suggesting that the brain provides long-range cues for other tissue systems during development. The presence of brain also protects embryos from otherwise-teratogenic agents. Overexpression of a hyperpolarization-activated cyclic nucleotide-gated ion channel rescues the muscle phenotype and the neural mispatterning that occur in brainless embryos, even when expressed far from the muscle or neural cells that mispattern. We identify a previously undescribed developmental role for the brain and reveal a non-local input into the control of early morphogenesis that is mediated by neurotransmitters and ion channel activity.Functions of the embryonic brain prior to regulating behavior are unclear. Here, the authors use an amputation assay in Xenopus laevis to demonstrate that removal of the brain early in development alters muscle and peripheral nerve patterning, which can be rescued by modulating bioelectric signals.

A Tunable Silk Hydrogel Device for Studying Limb Regeneration in Adult Xenopus Laevis.

In certain amphibian models limb regeneration can be promoted or inhibited by the local wound bed environment. This research introduces a device that can be utilized as an experimental tool to characterize the conditions that promotes limb regeneration in the adult frog (Xenopus laevis) model. In particular, this device was designed to manipulate the local wound environment via a hydrogel insert. Initial characterization of the hydrogel insert revealed that this interaction had a significant influence on mechanical forces to the animal, due to the contraction of the hydrogel. The material and mechanical properties of the hydrogel insert were a factor in the device design in relation to the comfort of the animal and the ability to effectively manipulate the amputation site. The tunable features of the hydrogel were important in determining the pro-regenerative effects in limb regeneration, which was measured by cartilage spike formation and quantified by micro-computed tomography. The hydrogel insert was a factor in the observed morphological outcomes following amputation. Future work will focus on characterizing and optimizing the device's observed capability to manipulate biological pathways that are essential for limb regeneration. However, the present work provides a framework for the role of a hydrogel in the device and a path forward for more systematic studies.

Update on stereology for light microscopy.

The quantitative investigation of images taken from light microscopy observation is one of the pillars of biological and biomedical investigation. The main objective is the count of objects, usually cells. In addition, the measurement of several morphological parameters, such as the diameter of cells, the length of vessels, etc., can also be important for the quantitative assessment of the features of a tissue. Whereas counting and measuring histological elements may appear easy, especially today with the availability of dedicated software, in fact it is not, since what we can count and measure on light microscopy images are not the true histological elements but actually profiles of them. Obviously, the number and size of profiles of an object do not correspond to the object number and size and thus significant mistakes can be made in the interpretation of the quantitative data obtained from profiles. To cope with this problem, over the last decades, a number of design-based stereological tools have been developed in order to obtain unbiased and reliable quantitative estimates of cell and tissue elements that originate from light microscopy images. This paper reviews the basic principles of the stereological tools from the first disector applications through some of the most recently devised methods.

Substitution of natural sensory input by artificial neurostimulation of an amputated trigeminal nerve does not prevent the degeneration of basal forebrain cholinergic circuits projecting to the somatosensory cortex.

Peripheral deafferentation downregulates acetylcholine (ACh) synthesis in sensory cortices. However, the responsible neural circuits and processes are not known. We irreversibly transected the rat infraorbital nerve and implanted neuroprosthetic microdevices for proximal stump stimulation, and assessed cytochrome-oxidase and choline- acetyl-transferase (ChAT) in somatosensory, auditory and visual cortices; estimated the number and density of ACh-neurons in the magnocellular basal nucleus (MBN); and localized down-regulated ACh-neurons in basal forebrain using retrograde labeling from deafferented cortices. Here we show that nerve transection, causes down regulation of MBN cholinergic neurons. Stimulation of the cut nerve reverses the metabolic decline but does not affect the decrease in cholinergic fibers in cortex or cholinergic neurons in basal forebrain. Artifical stimulation of the nerve also has no affect of ACh-innervation of other cortices. Cortical ChAT depletion is due to loss of corticopetal MBN ChAT-expressing neurons. MBN ChAT downregulation is not due to a decrease of afferent activity or to a failure of trophic support. Basalocortical ACh circuits are sensory specific, ACh is provided to each sensory cortex "on demand" by dedicated circuits. Our data support the existence of a modality-specific cortex-MBN-cortex circuit for cognitive information processing.

Chronic electrical stimulation of transected peripheral nerves preserves anatomy and function in the primary somatosensory cortex.

The structure and function of the central nervous system strongly depend on the organization and efficacy of the incoming sensory input. A disruption of somesthetic input severely alters the metabolic activity, electrophysiological properties and even gross anatomical features of the primary somatosensory cortex. Here we examined, in the rat somatosensory cortex, the neuroprotective and therapeutic effects of artificial sensory stimulation after irreversible unilateral transection of a peripheral sensory nerve (the infraorbital branch of the trigeminal nerve). The proximal stump of the nerve was inserted into a silicon tube with stimulating electrodes, through which continuous electrical stimulation was applied for 12 h/day (square pulses of 100 µs, 3.0 V, at 20 Hz) for 4 weeks. Deafferented animals showed significant decreases in cortical evoked potentials, cytochrome oxidase staining intensity (layers II-IV), cortical volume (layer IV) and number of parvalbumin-expressing (layers II-IV) and calbindin-D28k-expressing (layers II/III) interneurons. These deafferentation-dependent effects were largely absent in the nerve-stimulated animals. Together, these results provide evidence that chronic electrical stimulation has a neuroprotective and preservative effect on the sensory cortex, and raise the possibility that, by controlling the physical parameters of an artificial sensory input to a sectioned peripheral nerve, chronically deafferented brain regions could be maintained at near-'normal' conditions. Our findings could be important for the design of sensory neuroprostheses and for therapeutic purposes in brain lesions or neural degenerative processes.

Combining BMI Stimulation and Mathematical Modeling for Acute Stroke Recovery and Neural Repair.

Rehabilitation is a neural plasticity-exploiting approach that forces undamaged neural circuits to undertake the functionality of other circuits damaged by stroke. It aims to partial restoration of the neural functions by circuit remodeling rather than by the regeneration of damaged circuits. The core hypothesis of the present paper is that - in stroke - brain machine interfaces (BMIs) can be designed to target neural repair instead of rehabilitation. To support this hypothesis we first review existing evidence on the role of endogenous or externally applied electric fields on all processes involved in CNS repair. We then describe our own results to illustrate the neuroprotective and neuroregenerative effects of BMI-electrical stimulation on sensory deprivation-related degenerative processes of the CNS. Finally, we discuss three of the crucial issues involved in the design of neural repair-oriented BMIs: when to stimulate, where to stimulate and - the particularly important but unsolved issue of - how to stimulate. We argue that optimal parameters for the electrical stimulation can be determined from studying and modeling the dynamics of the electric fields that naturally emerge at the central and peripheral nervous system during spontaneous healing in both, experimental animals and human patients. We conclude that a closed-loop BMI that defines the optimal stimulation parameters from a priori developed experimental models of the dynamics of spontaneous repair and the on-line monitoring of neural activity might place BMIs as an alternative or complement to stem-cell transplantation or pharmacological approaches, intensively pursued nowadays.

Structural preservation of deafferented cortex induced by electrical stimulation of a sensory peripheral nerve.

Any manipulation to natural sensory input has direct effects on the morphology and physiology of the Central Nervous System. In the particular case of amputations, sensory areas of the brain undergo degenerative processes with a marked reduction in neuronal activity and global disinhibition. This is probably due to a deregulation of the circuits devoted to the control of the cortical activity. These changes are detected in the organization of the representational maps, the metabolic labeling by 2-deoxyglucose or cytochrome oxidase, the density of afferent and efferent axonal connections and the reduced expression of inhibitory neurotransmitters. In the present study, performed in animals, we have evaluated the therapeutic potential of Brain Machine Interfaces in reversing or limiting the degenerative/deregulation processes of amputations. Applying electrical stimulation on amputated peripheral nerves, we have achieved to maintain in approximately normal values 1) the cortical activity and 2) the expression of GABA-associated molecules of the inhibitory interneurons of the primary somatosensory cortex.