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.

The intrinsic laryngeal muscles are differentially modulated during respiration as well as other states and behaviors such as hypocapnia and sleep. Previous anatomical and pharmacological studies indicate a role for acetylcholine at the level of the nucleus ambiguus in the modulation of laryngeal motoneuron (LMN) activity. The present study investigated the anatomical nature of cholinergic input to inspiratory- (ILM) and expiratory-modulated (ELM) laryngeal motoneurons in the loose formation of the nucleus ambiguus. Using combined in vivo intracellular recording, dye filling, and immunohistochemistry, we demonstrate that LMNs identified in Sprague-Dawley rat receive several close appositions from vesicular acetylcholine transporter-immunoreactive (VAChT-ir) boutons. ELMs receive a significantly greater number of close appositions (mean ± standard deviation [SD]: 47 ± 11; n = 5) than ILMs (32 ± 9; n = 8; t-test P < 0.05). For both LMN types, more close appositions were observed on the cell soma and proximal dendrites compared to distal dendrites (two-way analysis of variance [ANOVA], P < 0.0001). Using fluorescence confocal microscopy, almost 90% of VAChT-ir close appositions (n = 45 boutons on n = 4 ELMs) were colocalized with the synaptic marker synaptophysin. These results support a strong influence of cholinergic input on LMNs and may have implications in the differential modulation of laryngeal muscle activity.

Respiratory modulation of sympathetic nerve activity (respSNA) was studied in a hypertensive rodent model of chronic kidney disease (CKD) using Lewis Polycystic Kidney (LPK) rats and Lewis controls. In adult animals under in vivo anaesthetised conditions (n = 8-10/strain), respiratory modulation of splanchnic and renal nerve activity was compared under control conditions, and during peripheral (hypoxia), and central, chemoreceptor (hypercapnia) challenge. RespSNA was increased in the LPK vs. Lewis (area under curve (AUC) splanchnic and renal: 8.7 ± 1.1 vs. 3.5 ± 0.5 and 10.6 ± 1.1 vs. 7.1 ± 0.2 μV.s, respectively, P < 0.05). Hypoxia and hypercapnia increased respSNA in both strains but the magnitude of the response was greater in LPK, particularly in response to hypoxia. In juvenile animals studied using a working heart brainstem preparation (n = 7-10/strain), increased respSNA was evident in the LPK (thoracic SNA, AUC: 0.86 ± 0.1 vs. 0.42 ± 0.1 μV.s, P < 0.05), and activation of peripheral chemoreceptors (NaCN) again drove a larger increase in respSNA in the LPK with no difference in the response to hypercapnia. Amplified respSNA occurs in CKD and may contribute to the development of hypertension.

Many respiration-related interneurons and motoneurons receive a catecholaminergic input, but the extent and distribution of this input to recurrent laryngeal motoneurons that innervate intrinsic muscles of the larynx are not clear. In the present study, we examined the catecholaminergic input to expiratory laryngeal motoneurons in the caudal nucleus ambiguus by combining intracellular labeling of single identified motoneurons, with immunohistochemistry to reveal tyrosine hydroxylase immunoreactive (catecholaminergic) terminal varicosities. Close appositions were found between the two structures, with 18 ± 5 close appositions per motoneuron (n = 7). Close appositions were more frequently observed on distal rather than proximal dendrites. Axosomatic appositions were not seen. In order to determine the source of this input, microinjections of cholera toxin B subunit (1%, 20 nl) were made into the caudal nucleus ambiguus. Retrogradely labeled neurons, located in the ipsilateral nucleus tractus solitarius and the area postrema, were tyrosine hydroxylase-positive. Our results not only demonstrate details of the extent and distribution of potential catecholamine inputs to the expiratory laryngeal motoneuron, but further indicate that the inputs, at least in part, originate from the dorsomedial medulla, providing a potential anatomical basis for previously reported catecholaminergic effects on the laryngeal adductor reflex.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.