Manipulating mitochondrial dynamics in the NTS prevents diet-induced deficits in brown fat morphology and glucose uptake.

AIMS: Brown adipose tissue (BAT) can produce heat by metabolizing glucose and fatty acids. Activation of BAT is controlled by the central nervous system (CNS) through sympathetic innervation. Dysregulation of signalling molecules in selective CNS areas such as the nucleus of tractus solitarius (NTS) are linked with altered BAT activity, obesity and diabetes. High-fat diet (HFD)-feeding increases mitochondrial fragmentation in the NTS, triggering insulin resistance, hyperphagia and weight gain. Here we sought to determine whether changes in mitochondrial dynamics in the NTS can affect BAT glucose uptake. MAIN METHODS: Rats received DVC stereotactic surgery for local brain administration of viruses that express mutated Drp1 genes. BAT glucose uptake was measured with PET/CT scans. Biochemical assays and immunohistochemistry determined altered levels of key signalling molecules and neural innervation of BAT. KEY FINDINGS: We show that short-term HFD-feeding decreases BAT glucose uptake. However, inhibiting mitochondrial fragmentation in NTS-astrocytes of HFD-fed rats partially restores BAT glucose uptake accompanied by lower blood glucose and insulin levels. Tyrosine Hydroxylase (TH) revealed that rats with inhibited mitochondrial fragmentation in NTS astrocytes had higher levels of catecholaminergic innervation in BAT compared to HFD-fed rats, and did not exhibit HFD-dependent infiltration of enlarged white fat droplets in the BAT. In regular chow-fed rats, increasing mitochondrial fragmentation in the NTS-astrocytes reduced BAT glucose uptake, TH immune-positive boutons and ß3-adrenergic receptor levels. SIGNIFICANCE: Our data suggest that targeting mitochondrial dynamics in the NTS-astrocytes could be a beneficial strategy to increase glucose utilization and protect from developing obesity and diabetes.

Graded spikes differentially signal neurotransmitter input in cerebrospinal fluid contacting neurons of the mouse spinal cord.

The action potential and its all-or-none nature is fundamental to neural communication. Canonically, the action potential is initiated once voltage-activated Na+ channels are activated, and their rapid kinetics of activation and inactivation give rise to the action potential's all-or-none nature. Here we demonstrate that cerebrospinal fluid contacting neurons (CSFcNs) surrounding the central canal of the mouse spinal cord employ a different strategy. Rather than using voltage-activated Na+ channels to generate binary spikes, CSFcNs use two different types of voltage-activated Ca2+ channel, enabling spikes of different amplitude. T-type Ca2+ channels generate small amplitude spikes, whereas larger amplitude spikes require high voltage-activated Cd2+-sensitive Ca2+ channels. We demonstrate that these different amplitude spikes can signal input from different transmitter systems; purinergic inputs evoke smaller T-type dependent spikes whereas cholinergic inputs evoke larger spikes that do not rely on T-type channels. Different synaptic inputs to CSFcNs can therefore be signaled by the spike amplitude.

GABAergic regulation of cell proliferation within the adult mouse spinal cord.

Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. We show that when ambient γ-aminobutyric acid (GABA) is increased in vigabatrin-treated or decreased by GAD67 allele haplodeficiency in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice of either sex, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2'-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signalling through specific sites on GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signalling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.

Kv3 Channels Contribute to the Excitability of Subpopulations of Spinal Cord Neurons in Lamina VII.

Autonomic parasympathetic preganglionic neurons (PGNs) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be because of recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within Lamina VII, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphologic analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast-firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Mediation of Cardiac Macrophage Activity via Auricular Vagal Nerve Stimulation Ameliorates Cardiac Ischemia/Reperfusion Injury.

BACKGROUND: Myocardial infarction (MI) reperfusion therapy causes paradoxical cardiac complications. Following restoration of blood flow to infarcted regions, a multitude of inflammatory cells are recruited to the site of injury for tissue repair. Continual progression of cardiac inflammatory responses does, however, lead to adverse cardiac remodeling, inevitably causing heart failure. MAIN BODY: Increasing evidence of the cardioprotective effects of both invasive and non-invasive vagal nerve stimulation (VNS) suggests that these may be feasible methods to treat myocardial ischemia/reperfusion injury via anti-inflammatory regulation. The mechanisms through which auricular VNS controls inflammation are yet to be explored. In this review, we discuss the potential of autonomic nervous system modulation, particularly via the parasympathetic branch, in ameliorating MI. Novel insights are provided about the activation of the cholinergic anti-inflammatory pathway on cardiac macrophages. Acetylcholine binding to the α7 nicotinic acetylcholine receptor (α7nAChR) expressed on macrophages polarizes the pro-inflammatory into anti-inflammatory subtypes. Activation of the α7nAChR stimulates the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This inhibits the secretion of pro-inflammatory cytokines, limiting ischemic injury in the myocardium and initiating efficient reparative mechanisms. We highlight recent developments in the controversial auricular vagal neuro-circuitry and how they may relate to activation of the cholinergic anti-inflammatory pathway. CONCLUSION: Emerging published data suggest that auricular VNS is an inexpensive healthcare modality, mediating the dynamic balance between pro- and anti-inflammatory responses in cardiac macrophages and ameliorating cardiac ischemia/reperfusion injury.

A versatile cholera toxin conjugate for neuronal targeting and tracing.

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.

Mechanisms underpinning sympathetic nervous activity and its modulation using transcutaneous vagus nerve stimulation.

NEW FINDINGS: What is the topic of this review? This review briefly considers what modulates sympathetic nerve activity and how it may change as we age or in pathological conditions. It then focuses on transcutaneous vagus nerve stimulation, a method of neuromodulation in autonomic cardiovascular control. What advances does it highlight? The review considers the pathways involved in eliciting the changes in autonomic balance seen with transcutaneous vagus nerve stimulation in relationship to other neuromodulatory techniques. The autonomic nervous system, consisting of the sympathetic and parasympathetic branches, is a major contributor to the maintenance of cardiovascular variables within homeostatic limits. As we age or in certain pathological conditions, the balance between the two branches changes such that sympathetic activity is more dominant, and this change in dominance is negatively correlated with prognosis in conditions such as heart failure. We have shown that non-invasive stimulation of the tragus of the ear increases parasympathetic activity and reduces sympathetic activity and that the extent of this effect is correlated with the baseline cardiovascular parameters of different subjects. The effects could be attributable to activation of the afferent branch of the vagus and, potentially, other sensory nerves in that region. This indicates that tragus stimulation may be a viable treatment in disorders where autonomic activity to the heart is compromised.

Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart-brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.-Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36-containing gap junctions.

Co-expression of GAD67 and choline acetyltransferase in neurons in the mouse spinal cord: A focus on lamina X.

Lamina X of the spinal cord is a functionally diverse area with roles in locomotion, autonomic control and processing of mechano and nociceptive information. It is also a neurochemically diverse region. However, the different populations of cells in lamina X remain to be fully characterised. To determine the co-localisation of the enzymes responsible for the production of GABA and acetylcholine (which play major roles in the spinal cord) in lamina X of the adult and juvenile mouse, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurons, combined with choline acetyltransferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurons were observed in lamina X of both adult and juvenile mice and in both age groups a population of cells containing both ChAT-IR and GAD67-GFP were observed in lumbar, thoracic and cervical spinal cord. Such dual labelled cells were predominantly located ventral to the central canal. Immunohistochemistry for vesicular acetylcholine transporter (VAChT) and GAD67 revealed a small number of double labelled terminals located lateral, dorsolateral and ventrolateral to the central canal. This study therefore describes in detail a population of ChAT-IR/GAD67-GFP neurons predominantly ventral to the central canal of the cervical, thoracic and lumbar spinal cord of adult and juvenile mice. These cells potentially correspond to a sub-population of the cholinergic central canal cluster cells which may play a unique role in controlling spinal cord circuitry.

The strange case of the ear and the heart: The auricular vagus nerve and its influence on cardiac control.

The human ear seems an unlikely candidate for therapies aimed at improving cardiac function, but the ear and the heart share a common connection: the vagus nerve. In recent years there has been increasing interest in the auricular branch of the vagus nerve (ABVN), a unique cutaneous subdivision of the vagus distributed to the external ear. Non-invasive electrical stimulation of this nerve through the skin may offer a simple, cost-effective alternative to the established method of vagus nerve stimulation (VNS), which requires a surgical procedure and has generated mixed results in a number of clinical trials for heart failure. This review discusses the available evidence in support of modulating cardiac activity using this strange auricular nerve.

Cholinergic Enhancement of Cell Proliferation in the Postnatal Neurogenic Niche of the Mammalian Spinal Cord.

The region surrounding the central canal (CC) of the spinal cord is a highly plastic area, defined as a postnatal neurogenic niche. Within this region are ependymal cells that can proliferate and differentiate to form new astrocytes and oligodendrocytes following injury and cerebrospinal fluid contacting cells (CSFcCs). The specific environmental conditions, including the modulation by neurotransmitters that influence these cells and their ability to proliferate, are unknown. Here, we show that acetylcholine promotes the proliferation of ependymal cells in mice under both in vitro and in vivo conditions. Using whole cell patch clamp in acute spinal cord slices, acetylcholine directly depolarized ependymal cells and CSFcCs. Antagonism by specific nicotinic acetylcholine receptor (nAChR) antagonists or potentiation by the α7 containing nAChR (α7*nAChR) modulator PNU 120596 revealed that both α7*nAChRs and non-α7*nAChRs mediated the cholinergic responses. Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) as a marker of cell proliferation, application of α7*nAChR modulators in spinal cord cultures or in vivo induced proliferation in the CC region, producing Sox-2 expressing ependymal cells. Proliferation also increased in the white and grey matter. PNU 120596 administration also increased the proportion of cells coexpressing oligodendrocyte markers. Thus, variation in the availability of acetylcholine can modulate the rate of proliferation of cells in the ependymal cell layer and white and grey matter through α7*nAChRs. This study highlights the need for further investigation into how neurotransmitters regulate the response of the spinal cord to injury or during aging.

Co-expression of GAD67 and choline acetyltransferase reveals a novel neuronal phenotype in the mouse medulla oblongata.

GABAergic and cholinergic systems play an important part in autonomic pathways. To determine the distribution of the enzymes responsible for the production of GABA and acetylcholine in areas involved in autonomic control in the mouse brainstem, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurones, combined with choline acetyl transferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurones were observed throughout the brainstem. A small number of cells contained both ChAT-IR and GAD67-GFP. Such double labelled cells were observed in the NTS (predominantly in the intermediate and central subnuclei), the area postrema, reticular formation and lateral paragigantocellular nucleus. All ChAT-IR neurones in the area postrema contained GAD67-GFP. Double labelled neurones were not observed in the dorsal vagal motor nucleus, nucleus ambiguus or hypoglossal nucleus. Double labelled ChAT-IR/GAD67-GFP cells in the NTS did not contain neuronal nitric oxide synthase (nNOS) immunoreactivity, whereas those in the reticular formation and lateral paragigantocellular nucleus did. The function of these small populations of double labelled cells is currently unknown, however their location suggests a potential role in integrating signals involved in oromotor behaviours.

Sympathetic preganglionic neurons: properties and inputs.

The sympathetic nervous system comprises one half of the autonomic nervous system and participates in maintaining homeostasis and enabling organisms to respond in an appropriate manner to perturbations in their environment, either internal or external. The sympathetic preganglionic neurons (SPNs) lie within the spinal cord and their axons traverse the ventral horn to exit in ventral roots where they form synapses onto postganglionic neurons. Thus, these neurons are the last point at which the central nervous system can exert an effect to enable changes in sympathetic outflow. This review considers the degree of complexity of sympathetic control occurring at the level of the spinal cord. The morphology and targets of SPNs illustrate the diversity within this group, as do their diverse intrinsic properties which reveal some functional significance of these properties. SPNs show high degrees of coupled activity, mediated through gap junctions, that enables rapid and coordinated responses; these gap junctions contribute to the rhythmic activity so critical to sympathetic outflow. The main inputs onto SPNs are considered; these comprise afferent, descending, and interneuronal influences that themselves enable functionally appropriate changes in SPN activity. The complexity of inputs is further demonstrated by the plethora of receptors that mediate the different responses in SPNs; their origins and effects are plentiful and diverse. Together these different inputs and the intrinsic and coupled activity of SPNs result in the rhythmic nature of sympathetic outflow from the spinal cord, which has a variety of frequencies that can be altered in different conditions.

How sympathetic are your spinal cord circuits?

NEW FINDINGS: What is the topic of this review? This review focuses on the role of gap junctions and interneurones in sympathetic control at the spinal cord level. What advances does it highlight? The review considers the importance of these local spinal circuits in contributing to rhythmic autonomic activity and enabling appropriate responses to homeostatic perturbations. Sympathetic control of end organs relies on the activity of sympathetic preganglionic neurones (SPNs) within the spinal cord. These SPNs exhibit heterogeneity with respect to function, neurochemistry, location, descending inputs and patterns of activity. Part of this heterogeneity is bestowed by local spinal circuitry. Our understanding of the role of these local circuits, including the significance of connections between the SPNs themselves through specialized gap junctions, is patchy. This report focuses on interneurones and gap junctions within these circuits. Gap junctions play a role in sympathetic control; they are located on SPNs in the intermediolateral cell column. Mefloquine, a chemical that blocks these gap junctions, reduces local rhythmic activity in the spinal cord slice and disrupts autonomic control in the working heart-brainstem preparation. The role that these gap junctions may play in health and disease in adult animals remains to be elucidated fully. Presympathetic interneurones are located in laminae V, VII and X and the intermediolateral cell column; those in lamina X are GABAergic and directly inhibit SPNs. The GABAergic inputs onto SPNs exert their effects through activation of synaptic and extrasynaptic receptors, which stabilize the membrane at negative potentials. The GABAergic interneurones contribute to rhythmic patterns of activity that can be generated in the spinal cord, because bicuculline reduces network oscillatory activity. These studies indicate that local spinal cord circuitry is critical in enabling appropriate levels and patterning of activity in sympathetic outflow. We need to understand how these circuits may be harnessed in the situation of spinal cord injury.

Non-invasive vagus nerve stimulation in healthy humans reduces sympathetic nerve activity.

BACKGROUND: Vagus nerve stimulation (VNS) is currently used to treat refractory epilepsy and is being investigated as a potential therapy for a range of conditions, including heart failure, tinnitus, obesity and Alzheimer's disease. However, the invasive nature and expense limits the use of VNS in patient populations and hinders the exploration of the mechanisms involved. OBJECTIVE: We investigated a non-invasive method of VNS through electrical stimulation of the auricular branch of the vagus nerve distributed to the skin of the ear--transcutaneous VNS (tVNS) and measured the autonomic effects. METHODS: The effects of tVNS parameters on autonomic function in 48 healthy participants were investigated using heart rate variability (HRV) and microneurography. tVNS was performed using a transcutaneous electrical nerve stimulation (TENS) machine and modified surface electrodes. Participants visited the laboratory once and received either active (200 µs, 30 Hz; n = 34) or sham (n = 14) stimulation. RESULTS: Active tVNS significantly increased HRV in healthy participants (P = 0.026) indicating a shift in cardiac autonomic function toward parasympathetic predominance. Microneurographic recordings revealed a significant decrease in frequency (P = 0.0001) and incidence (P = 0.0002) of muscle sympathetic nerve activity during tVNS. CONCLUSION: tVNS can increase HRV and reduce sympathetic nerve outflow, which is desirable in conditions characterized by enhanced sympathetic nerve activity, such as heart failure. tVNS can therefore influence human physiology and provide a simple and inexpensive alternative to invasive VNS.

Anodal transcranial direct current stimulation (tDCS) over the motor cortex increases sympathetic nerve activity.

BACKGROUND: Transcranial direct current stimulation (tDCS) is currently being investigated as a non-invasive neuromodulation therapy for a range of conditions including stroke rehabilitation. tDCS affects not only the area underlying the electrodes but also other areas of the cortex and subcortical structures. This could lead to unintended alteration in brain functions such as autonomic control. OBJECTIVE: We investigated the potential effects of tDCS on cardiovascular autonomic function in healthy volunteers. METHODS: Anodal (n = 14) or cathodal (n = 8) tDCS at 1 mA was applied over the primary motor cortex with the second electrode placed on the contralateral supraorbital region. Subjects visited the department twice and received active or sham tDCS for 15 min. Heart rate, blood pressure and respiration were recorded at baseline, during tDCS and after stimulation. Heart rate variability (HRV) was calculated using spectral analysis of beat-to-beat intervals derived from ECG data. Microneurography was also used to record muscle sympathetic nerve activity (MSNA; n = 5). RESULTS: Anodal tDCS caused a significant shift in HRV toward sympathetic predominance (P = 0.017), whereas there was no significant change in the cathodal or sham groups. Microneurography results also showed a significant increase in MSNA during anodal tDCS that continued post-stimulation. CONCLUSIONS: Anodal tDCS of the motor cortex shifts autonomic nervous system balance toward sympathetic dominance due at least in part to an increase in sympathetic output. These results suggest further investigation is warranted on tDCS use in patient groups with potential autonomic dysfunction, such as stroke patients.

GABAergic responses of mammalian ependymal cells in the central canal neurogenic niche of the postnatal spinal cord.

The area surrounding the central canal of the postnatal mammalian spinal cord is a highly plastic region that exhibits many similarities to other postnatal neurogenic niches, such as the subventricular zone. Within this region, ependymal cells have been identified as neural stem cells however very little is known about their properties and how the local environment, including neurotransmitters, is capable of affecting them. The neurotransmitter GABA is present around the central canal and is known to affect cells within other postnatal neurogenic niches. This study used whole cell patch clamp electrophysiology and intracellular dye-loading in in vitro Wistar rat spinal cord slices to characterise ependymal cells and their ability to respond to GABA. Ependymal cells were defined by their passive response properties and low input resistances. Extensive dye-coupling was observed between ependymal cells; this was confirmed as gap junction coupling using the gap junction blocker, 18ß-glycyrrhetinic acid, which significantly increased the input resistance of ependymal cells. GABA depolarised all ependymal cells tested; the partial antagonism of this response by bicuculline and gabazine indicates that GABA(A) receptors contribute to this response. A lack of effect by baclofen suggests that GABA(B) receptors do not contribute to the GABAergic response. The ability of ependymal cells to respond to GABA suggests that GABA could be capable of influencing the proliferation and differentiation of cells within the neurogenic niche of the postnatal spinal cord.

A simple method to fluorescently label pericytes in the CNS and skeletal muscle.

Pericytes play important roles in vascular control and may form an important part of the blood brain barrier. Here we introduce a simple method for fluorescently labelling pericytes to enable further studies in live or fixed tissue of rats and mice. Following intraperitoneal injection, the fluorescent tracer Fluorogold was rapidly taken up into vascular endothelial cells, and within 3h in the central nervous system appeared within small perivascular cells with a morphology consistent with pericytes. These Fluorogold labelled cells were pericytes since they displayed immunoreactivity for platelet derived growth factor receptor ß and were closely associated with isolectin B4 binding to endothelial cells. Pericytes in skeletal muscle were also labelled with this method, but not those within the heart, lungs or kidney. This simple method could therefore be applied for labelling pericytes in a wide variety of studies, including live cell imaging or immunohistochemistry.