A brainstem preparation allowing simultaneous access to respiratory motor output and cellular properties of motoneurons in American bullfrogs

Abstract

Breathing is generated by a complex neural circuit, and the ability to monitor the activity of multiple network components simultaneously is required to uncover the cellular basis of breathing. In neonatal rodents, a single brainstem slice can be produced to record respiratory-related motor nerve discharge along with individual rhythm generating cells or motoneurons due to the close proximity of these neurons in the brainstem. However, most ex vivo preparations in other vertebrates can only capture respiratory motor outflows or electrophysiological properties of putative respiratory neurons in slices without relevant synaptic inputs. Here, we detail a method to horizontally slice away the dorsal portion of the brainstem to expose fluorescently-labeled motoneurons for patch-clamp recordings in American bullfrogs. This “semi-intact” preparation allows tandem recordings of motor output and single motoneurons during respiratory-related synaptic inputs. The rhythmic motor patterns are comparable to intact preparations and operate at physiological temperature and [K+]. Thus, this preparation provides the ability to record network and cellular-level outputs simultaneously and may lead to new mechanistic insights into breathing control across vertebrates.

Neuron populations use variable combinations of short-term feedback mechanisms to stabilize firing rate

“…We last sought to determine if respiratory motoneurons experience slow activity-dependent hyperpolarization in the intact network, and then, if reciprocal co-expression of the dynamic Na + pump and Kv7 channels plays a role in regulating firing rate during realistic activity. Fig 4A shows whole-cell current clamp recordings of identified vagal motoneurons from 3 different “semi-intact preparations” receiving physiological synaptic inputs from the respiratory rhythm generator, as described in reference [ 45 ]. After rhythmic bursts associated with lung ventilation, each neuron exhibited an afterhyperpolarization that lasted for several seconds before the next burst.…”

“…We then loaded the fourth branch of vagus nerve with a fluorescent dye (Dextran, Tetramethylrhodamine, 3000 MW, anionic) for 1 hour on 1 side and 2 hours on the other (3 hours total), as approximately 75% of these neurons receive respiratory-related synaptic input associated with breathing [ 45 ]. Transverse 300 μm-thick slices of the brainstem (for brainstem slices) or the dorsal portion of the brainstem (for the “semi-intact” network preparation) [ 45 ] were then cut using a Vibratome Series 1000 sectioning system in ice-cold aCSF bubbled with 98.5% O 2 /1.5% CO 2 . Tissue preparations were given 1 hour to recover and maintained in room temperature aCSF bubbled with 98.5% O 2 /1.5% CO 2 throughout the experiments.…”

“…To determine whether slow hyperpolarizations follow burst firing in the intact respiratory network, we used a semi-intact preparation that allows simultaneous access to extracellular nerve root recordings and motoneuron patch clamp recordings during the ongoing respiratory rhythm [ 45 ]. We also used this preparation to make recordings of synaptic currents onto the motoneuron that are associated with breathing.…”

“…For this, we constructed a current clamp protocol that injected cells with current that matched the exact waveform of synaptic input recorded from the intact network and paced them at a frequency consistent with the respiratory rhythm. Thus, we could restore breathing-related motor patterns in neurons that were disconnected from the rhythmic network [ 22 , 45 ]. The protocol consisted of 10, approximately 1-second injections of physiological synaptic input separated by an approximately 9-second-long interval.…”

“…"semi-intact" network preparation) [45] were then cut using a Vibratome Series 1000 sectioning system in ice-cold aCSF bubbled with 98.5% O 2 /1.5% CO 2 . Tissue preparations were given 1 hour to recover and maintained in room temperature aCSF bubbled with 98.5% O 2 /1.5% CO 2 throughout the experiments.…”