Three brainstem areas involved in respiratory rhythm generation in bullfrogs

Abstract

For most multiphasic motor patterns, rhythm and pattern are produced by the same circuit elements. For respiration, however, these functions have long been assumed to occur separately. In frogs, the ventilatory motor pattern produced by the isolated brainstem consists of buccal and biphasic lung bursts. Previously, two discrete necessary and sufficient sites for lung and buccal bursts were identified. Here we identify a third site, the Priming Area, important for and having neuronal activity correlated with the first phase of biphasic lung bursts. As each site is important for burst generation of a separate phase, we suggest each major phase of ventilation is produced by an anatomically distinct part of an extensive brainstem network. Embedding of discrete circuit elements producing major phases of respiration within an extensive rhythmogenic brainstem network may be a shared architectural characteristic of vertebrates.

Abstract: Ventilation in mammals consists of at least three distinct phases: inspiration, post-inspiration and late-expiration. While distinct brainstem rhythm generating and pattern forming networks have long been assumed, recent data suggest the mammalian brainstem contains two coupled neuronal oscillators: one for inspiration and the other for active expiration. However, whether additional burst generating ability is required for generating other phases of ventilation in mammals is controversial. To investigate brainstem circuit architectures capable of producing multiphasic ventilatory rhythms, we utilized the isolated frog brainstem. This preparation produces two types of ventilatory motor patterns, buccal and lung bursts. Lung bursts can be divided into two phases, priming and powerstroke. Previously we identified two putative oscillators, the Buccal and Lung Areas. The Lung Area produces the lung powerstroke and the Buccal Area produces buccal bursts and - we assumed - the priming phase of lung bursts. However, here we identify an additional brainstem region that generates the priming phase. This Priming Area extends rostral and caudal of the Lung Area and is distinct from the Buccal Area. Using AMPA microinjections and reversible synaptic blockade, we demonstrate selective excitation and ablation (respectively) of priming phase activity. We also demonstrate that the Priming Area contains neurons active selectively during the priming phase. Thus, we propose that three distinct neuronal components generate the multiphase respiratory motor pattern produced by the frog brainstem: the buccal, priming and powerstroke burst generators. This raises the possibility that a similar multi-burst generator architecture mediates the three distinct phases of ventilation in mammals.

Figure 1. Schematic diagram of the sheep dip experimental technique

A, the isolated frog brainstem is mounted vertically onto a platform and superfused with (2% CO₂ and 98% O₂ equilibrated) normal saline via a constant drip from above. A cylindrical chamber is placed beneath the vertical mount and is filled with 20–40 mm Mg²⁺ saline. The chamber can be moved vertically to increase or decrease the amount of brainstem exposed to the high-Mg²⁺ solution. B, recordings showing representative activity in CNV, CNVII, CNX and CNXII of the vertically mounted brainstem before exposure to high-Mg²⁺ solution. CN, cranial nerve. Red sections illustrates lung priming phase activity. (Modified from Duchcherer et al. (2013).)

Figure 2. Example of the systematic survey in caudal to rostral direction

Respiratory activity in an isolated frog brainstem under control conditions included buccal (B) and two-phase lung burst patterns (top traces: L1, priming; L2, powerstroke). When the preparation was submerged in high-Mg²⁺ up to Level A, buccal activity was abolished while biphasic lung bursts persisted. Submersion to Level B caused cessation of the priming phase but not the powerstroke phase of lung bursts. Submersion to Level C abolished all ventilatory motor patterns. CN, cranial nerves. n = 10.

Figure 3. Example of the systematic survey in rostral to caudal direction

Respiratory activity in an isolated brainstem under control conditions included buccal (B) and biphasic lung burst patterns (top traces: L1, priming; L2, powerstroke). When the preparation was submerged in high-Mg²⁺ solution up to Level X the first priming phase of lung bursts was lost but the second powerstroke phase and buccal bursts persisted. Further submersion to Level Y abolished lung activity entirely, but buccal activity persisted. CN, cranial nerve. n = 7.

Figure 4. Buccal and lung burst frequencies during the systematic surveys

A, caudal to rostral survey. Left panel displays buccal burst frequency over increasing brainstem submersion levels (A, B and C correspond to Levels A, B and C in Fig. 2). Right panel displays lung burst frequency. B, rostral to caudal survey. Left panel displays buccal burst frequency (x and y correspond to Levels X and Y in Fig. 3). Right panel displays lung burst frequency.

Figure 5. AMPA microinjections around the Lung Area cause reversible increases in priming phase activity

A, location of rostral, central and caudal AMPA microinjections sites displayed relative to the cranial nerves. Top panel shows the location of microinjections relative to CNVI. Individual crosses represent the data points, the large plus sign represents their average and the shaded (blue) circle represents the estimated size of the sphere formed by the microinjections. Bottom panel displays the depth of the injections. Crosses represent topographically identified locations, and circles represent post hoc analysis of dye injection into the region of interest. B, representative traces from CNXII from all three sites showing reversible increases in priming phase activity. C, group data displaying increases in priming activity (per unit lung) following AMPA microinjection. CN, cranial nerve. n = 16.

Figure 6. Extracellular units recorded from the three injection sites selectively fire during the priming phase

A, sample traces of extracellular unit recordings (Ext). B, raster plot of extracellular units that fired during priming and powerstroke phases of the lung breath. C, group data displaying the proportion of units recorded from each injection area. CN, cranial nerve. ^*, the caudal site contains significantly more priming units compared to the other two sites. n = 19.

Figure 7. Summary schematic of the respiratory centres in the frog brainstem

Ventral brainstem displayed with rostral up. Priming Area in red (r4–r6), Lung Area in blue (r5) and Buccal Area in green (r7–r8). The grey lines and r-labels represent the rhombomeric segments of the brainstem (Straka et al. 2006).