Somatostatin in the rat rostral ventrolateral medulla: Origins and mechanism of action.

Somatostatin (SST) or agonists of the SST-2 receptor (sst2 ) in the rostral ventrolateral medulla (RVLM) lower sympathetic nerve activity, arterial pressure, and heart rate, or when administered within the Bötzinger region, evoke apneusis. Our aims were to describe the mechanisms responsible for the sympathoinhibitory effects of SST on bulbospinal neurons and to identify likely sources of RVLM SST release. Patch clamp recordings were made from bulbospinal RVLM neurons (n = 31) in brainstem slices prepared from juvenile rat pups. Overall, 58% of neurons responded to SST, displaying an increase in conductance that reversed at -93 mV, indicative of an inwardly rectifying potassium channel (GIRK) mechanism. Blockade of sst2 abolished this effect, but application of tetrodotoxin did not, indicating that the SST effect is independent of presynaptic activity. Fourteen bulbospinal RVLM neurons were recovered for immunohistochemistry; nine were SST-insensitive and did not express sst2a . Three out of five responsive neurons were sst2a -immunoreactive. Neurons that contained preprosomatostatin mRNA and cholera-toxin-B retrogradely transported from the RVLM were detected in: paratrigeminal nucleus, lateral parabrachial nucleus, Kölliker-Fuse nucleus, ventrolateral periaqueductal gray area, central nucleus of the amygdala, sublenticular extended amygdala, interstitial nucleus of the posterior limb of the anterior commissure nucleus, and bed nucleus of the stria terminalis. Thus, those brain regions are putative sources of endogenous SST release that, when activated, may evoke sympathoinhibitory effects via interactions with subsets of sympathetic premotor neurons that express sst2 .

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.

Orexinergic activation of medullary premotor neurons modulates the adrenal sympathoexcitation to hypothalamic glucoprivation.

Glucoprivation activates neurons in the perifornical hypothalamus (PeH) and in the rostral ventrolateral medulla (RVLM), which results in the release of adrenaline. The current study aimed to establish 1) whether neuroglucoprivation in the PeH or in the RVLM elicits adrenaline release in vivo and 2) whether direct activation by glucoprivation or orexin release in the RVLM modulates the adrenaline release. Neuroglucoprivation in the PeH or RVLM was elicited by microinjections of 2-deoxy-D-glucose or 5-thio-D-glucose in anesthetized, euglycemic rats. Firstly, inhibition of neurons in the PeH abolished the increase in adrenal sympathetic nerve activity (ASNA) to systemic glucoprivation. Secondly, glucoprivation of neurons in the PeH increased ASNA. Thirdly, in vivo or in vitro glucoprivation did not affect the activity of RVLM adrenal premotor neurons. Finally, blockade of orexin receptors in the RVLM abolished the increase in ASNA to neuroglucoprivation in the PeH. The evoked changes in ASNA were directly correlated to levels of plasma metanephrine but not to normetanephrine. These findings suggest that orexin release modulates the activation of adrenal presympathetic neurons in the RVLM.