Susan A. Deuchars

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. Here we show that when ambient GABA is increased in vigabatrin-treated or decreased in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice, 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 the possibility of 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 the 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 signaling through specific sites on the GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signaling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.

Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological 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. Significance statement Neural circuits in the spinal cord and pons mediate the micturition reflex. The spinal cord drives bladder contraction during micturition through the activation of parasympathetic preganglionic neurons in lamina VII of the lumbosacral spinal cord. Despite the significant contribution of these neurons to a crucial physiological reflex, neurons in this region have been under-characterised. This study therefore elucidated and thoroughly characterised distinct neuronal populations in this lamina; we propose that these populations included a fast firing interneuron and subtypes of parasympathetic preganglionic neurons. Further investigation revealed the critical importance of Kv3 channels in the fast firing ability of the interneurons, as well as in synaptic release onto parasympathetic preganglionic neurons.

The action potential and its all-or-none nature is fundamental to neural communication. Canonically the action potential is initiated once voltage-gated 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 show that cerebrospinal fluid contacting neurons (CSFcNs) surrounding the central canal of the mouse spinal cord employ a different strategy. Rather than using voltage-gated Na ⁺ channels to generate binary spikes, CSFcNs use two different types of voltage-gated Ca ²⁺ channel, enabling spikes of different amplitude. T-type Ca ²⁺ channels generate small amplitude spikes, whereas large amplitude spikes require high voltage-activated Cd ²⁺ sensitive Ca ²⁺ channels. We show that these different amplitude spikes signal input from different transmitter systems; purinergic inputs evoke smaller T-type dependent spikes while cholinergic inputs evoke large T-type independent spikes. Different synaptic inputs to CSFcNs can therefore be signalled by the spike amplitude.

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.

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.

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. FASEB J. 31, 3966–3977 (2017). www.fasebj.org —Lall, Varinder K., Bruce, Gareth, Voytenko, Larysa, Drinkhill, Mark, Wellershaus, Kerstin, Willecke, Klaus, Deuchars, Jim, Deuchars, Susan A. Physiologie regulation of heart rate and blood pressure involves connexin 36–containing gap junctions. FASEB J. 31, 3966–3977 (2017)