Anita Turner

Mapping and analysis of the connectome of sympathetic premotor neurons in the rostral ventrolateral medulla of the rat using a volumetric brain atlas

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88–94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data.

Rostroventrolateral medulla neurons with commissural projections provide input to sympathetic premotor neurons: anatomical and functional evidence

The activity of neurons in the rostral ventrolateral medulla (RVLM) is critical for the generation of vasomotor sympathetic tone. Multiple pre‐sympathetic pathways converge on spinally projecting RVLM neurons, but the origin and circumstances in which such inputs are active are poorly understood. We have previously shown that input from the contralateral brainstem contributes to the baseline activity of this population: in the current study we investigate the distribution, phenotype and functional properties of RVLM neurons with commissural projections in the rat. We firstly used retrograde transport of fluorescent microspheres to identify neurons that project to the contralateral RVLM. Labelled neurons were prominent in a longitudinal column that extended over 1 mm caudal from the facial nucleus and contained hybridisation products indicating enkephalin (27%), GABA (15%) and adrenaline (3%) synthesis and included 6% of bulbospinal neurons identified by transport of cholera toxin B. Anterograde transport of fluorescent dextran‐conjugate from the contralateral RVLM revealed extensive inputs throughout the RVLM that frequently terminated in close apposition with catecholaminergic and bulbospinal neurons. In urethane‐anaesthetised rats we verified that 28/37 neurons antidromically activated by electrical stimulation of the contralateral pressor region were spontaneously active, of which 13 had activity locked to central respiratory drive and 15 displayed ongoing tonic discharge. In six tonically active neurons sympathoexcitatory roles were indicated by spike‐triggered averages of splanchnic sympathetic nerve activity. We conclude that neurons in the RVLM project to the contralateral brainstem, form synapses with sympathetic premotor neurons, and have functional properties consistent with sympthoexcitatory function.

Recording, labeling, and transfection of single neurons in deep brain structures

Genetic tools that permit functional or connectomic analysis of neuronal circuits are rapidly transforming neuroscience. The key to deployment of such tools is selective transfection of target neurons, but to date this has largely been achieved using transgenic animals or viral vectors that transduce subpopulations of cells chosen according to anatomical rather than functional criteria. Here, we combine single‐cell transfection with conventional electrophysiological recording techniques, resulting in three novel protocols that can be used for reliable delivery of conventional dyes or genetic material in vitro and in vivo. We report that techniques based on single cell electroporation yield reproducible transfection in vitro, and offer a simple, rapid and reliable alternative to established dye‐labeling techniques in vivo, but are incompatible with targeted transfection in deep brain structures. In contrast, we show that intracellular electrophoresis of plasmid DNA transfects brainstem neurons recorded up to 9 mm deep in the anesthetized rat. The protocols presented here require minimal, if any, modification to recording hardware, take seconds to deploy, and yield high recovery rates in vitro (dye labeling: 89%, plasmid transfection: 49%) and in vivo (dye labeling: 66%, plasmid transfection: 27%). They offer improved simplicity compared to the juxtacellular labeling technique and for the first time offer genetic manipulation of functionally characterized neurons in previously inaccessible brain regions.

DBA/2J mouse model for experimental glaucoma: pitfalls and problems

The DBA/2J mouse has been described as a model for congenital experimental glaucoma. It develops anterior segment anomalies with synechiae and pigment dispersion leading to raised intraocular pressure and glaucomatous damage. However, there are serious practical considerations when using this model in longitudinal studies.

Somatostatin 2a receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column of the rat

Microinjection of somatostatin (SST) causes site‐specific effects on respiratory phase transition, frequency, and amplitude when microinjected into the ventrolateral medulla (VLM) of the anesthetized rat, suggesting selective expression of SST receptors on different functional classes of respiratory neurons. Of the six subtypes of SST receptor, somatostatin 2a (sst2a) is the most prevalent in the VLM, and other investigators have suggested that glutamatergic neurons in the preBötzinger Complex (preBötC) that coexpress neurokinin‐1 receptor (NK1R), SST, and sst2a are critical for the generation of respiratory rhythm. However, quantitative data describing the distribution of sst2a in respiratory compartments other than preBötC, or on functionally identified respiratory neurons, is absent. Here we examine the medullary expression of sst2a with particular reference to glycinergic/expiratory neurons in the Bötzinger Complex (BötC) and NK1R‐immunoreactive/inspiratory neurons in the preBötC. We found robust sst2a expression at all rostrocaudal levels of the VLM, including a large proportion of catecholaminergic neurons, but no colocalization of sst2a and glycine transporter 2 mRNA in the BötC. In the preBötC 54% of sst2a‐immunoreactive neurons were also positive for NK1R. sst2a was not observed in any of 52 dye‐labeled respiratory interneurons, including seven BötC expiratory‐decrementing and 11 preBötC preinspiratory neurons. We conclude that sst2a is not expressed on BötC respiratory neurons and that phasic respiratory activity is a poor predictor of sst2a expression in the preBötC. Therefore, sst2a is unlikely to underlie responses to BötC SST injection, and is sparse or absent on respiratory neurons identified by classical functional criteria. J. Comp. Neurol. 524:1384–1398, 2016.

Somatostatin 2A receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column

This free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, AustraliaThis free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, Australia

The connectome of rostral ventrolateral medulla sympathetic premotor neurons

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are thought to play a critical role in the generation of vasomotor sympathetic tone and represent a major site of convergence for multiple descending and reflex pathways that co-ordinate sympathetic nerve activity. Elucidating the organization of the circuits that drive these neurons is a key research objective. Here we present brain-wide connectomic maps of neurons that provide monosynaptic drive to putative RVLM sympathetic premotor neurons, generated using a two-step restricted trans-synaptic viral tracing strategy. We made focal microinjections of a genetically restricted reporter-expressing rabies vector, SADΔG(EnvA)-RFP, into the RVLM, and restricted its entry to neurons that project to the T2 spinal cord by first transducing them with a cassette required for rabies entry and trans-synapsis, YTB. By precisely targeting rabies injections to the core of the RVLM bulbospinal population we were able to restrict rabies ‘seeding’ to small numbers (as low as one single neuron) of RVLM presympathetic neurons immediately caudal to the facial nucleus, and then map the locations of neurons that provide monosynaptic input to the seed population. We observed reproducible patterns of inputs arising from the dorsal, contralateral, and midline medulla, and local RVLM interneurons including catecholaminergic non-bulbospinal neurons and neurons likely to reside within the ventral respiratory column. Distant inputs were identified in the pons, cerebellum and midbrain, and included previously suspected sites of monosynaptic drive such as the paraventricular nucleus of the hypothalamus.