Evidence of intermediate reticular formation involvement in swallow pattern generation, recorded optically in the neonate rat sagittally sectioned hindbrain

Abstract

Swallow is a primitive behavior regulated by medullary networks, responsible for movement of food/liquid from the oral cavity to the esophagus. To investigate how functionally heterogeneous networks along the medullary intermediate reticular formation (IRt) and ventral respiratory column (VRC) control swallow, we electrically stimulated the nucleus tractus solitarius to induce fictive swallow between inspiratory bursts, with concurrent optical recordings using a synthetic Ca²⁺ indicator in the neonatal sagittally sectioned rat hindbrain (SSRH) preparation. Simultaneous recordings from hypoglossal nerve rootlet (XIIn) and ventral cervical spinal root C1-C2 enabled identification of the system-level correlates of 1) swallow (identified as activation of the XIIn but not the cervical root) and 2) Breuer-Hering expiratory reflex (BHE; lengthened expiration in response to stimuli during expiration). Optical recording revealed reconfiguration of respiration-modulated networks in the ventrolateral medulla during swallow and the BHE reflex. Recordings identified novel spatially compact networks in the IRt near the facial nucleus (VIIn) that were active during fictive swallow, suggesting that the swallow network is not restricted to the caudal medulla. These findings also establish the utility of using this in vitro preparation to investigate how functionally heterogeneous medullary networks interact and reconfigure to enable a repertoire of orofacial behaviors.NEW & NOTEWORTHY For the first time, medullary networks that control breathing and swallow are recorded optically. Episodic swallows are induced via electrical stimulation along the dorsal medulla, in and near the NTS, during spontaneously occurring fictive respiration. These findings establish that networks regulating both orofacial behaviors and breathing are accessible for optical recording at the surface of the sagittally sectioned rodent hindbrain preparation.

Keywords: Ca2+ imaging; medulla; rat; respiration; swallow.

Graphical abstract

Figure 1.

Neurons activated during fictive swallow are interdigitated with respiration modulated neurons along the ventral respiratory column. A: cartoon of the preparation. Left panel shows the tilted sagittal face of the preparation, which tapers medial toward its caudal margin and tapers lateral toward its ventral margin (black triangles dm, vl), with functional anatomical landmarks added. BötC, Bötzinger complex; cNTS, caudal nucleus tractus solitarii; NA, nucleus ambiguus; VIIn, facial nucleus; VRC, ventral respiratory column). The vertical line through VIIn indicates the level of section of the transverse plane cartoon (right); the yellow diagonal line in the transverse view indicates the surface exposed for optical recording. B: traces generated by respiratory neurons (top traces, aligned to C2 motor output), and neurons activated during fictive swallow (bottom traces, aligned to fictive swallow bursts in XII motor output). C: a tiled image of one preparation, showing stimulus location (blue square), optical recording field of view (bordered by a broken yellow line) and location of stimulus-activated neurons (red dots) and respiration-modulated neurons (blue dots). The VIIn is outlined in a broken black line. D: location of the center of mass of the ROI generated by somatic Ca²⁺ transients associated with swallow-activated (red crosses, n = 65) and respiration-modulated neurons (blue dots, n = 34), in and dorsal to the VRC, pooled from 3 experiments, aligned to VIIc (x-axis), and ventral surface (y-axis).

Figure 2.

IRt networks at the dorsal edge of, and dorsal to, the facial nucleus are activated during stimulus-induced fictive swallow. A: tiled image of representative preparation, showing stimulus location (blue square), optical recording field of view (bordered by a broken yellow line), and location of ROIs from which traces were extracted (red dots). Bi: motor output (XII, C2) and optical recordings during four fictive swallows. Stimuli evoked XII bursts, but not C2 bursts. High-pass-filtered optical traces of 136 somata are sorted from rapidly to slowly adapting units. Bii: stimulus-triggered averages (STAs) of traces shown in Bi highlight variability in neuronal responses. At the top are superimposed stimulus-triggered averages of the most rapidly (red trace) and second-most slowly adapting (green trace) neurons. C: histogram of normalized STA half-widths reveals a skewed distribution, with a small number of slowly adapting neurons. D: pooled data from three experiments, aligned to VIIc (x-axis) and ventral surface (y-axis; n = 394 neurons), color coded from fast- (0.0–0.39, AU) to slow-adapting (1.0, AU) does not reveal any parcellation of slowly adapting neurons. Each point represents the center of mass of the ROI generated by somatic Ca²⁺ transients. Histograms (inset) of cell numbers reveal a bimodal distribution in the ventrodorsal axis, suggesting the existence of two distinct networks, one centered at the dorsal margin of VIIn (peak at 400 μm from the ventral surface) and the other centered at 800 μm from the ventral surface. At the sampling rates used here, no differences in onset times or pattern of activity between groups could be detected. AU, arbitrary units; IRt, intermediate reticular formation; ROI, region of interest.

Figure 3.

Identification of regions along dorsal and midbrainstem capable of evoking swallow, the Breuer–Hering expiratory reflex (BHE), and respiratory entrainment. A: tiled image of one preparation with symbols indicating stimulus location. B: effects of stimuli at the locations indicated in A. Bi: swallow with lengthened expiration (white square). Bii: swallow with shortened expiration (black triangle). Biii: swallow that had no effect on expiratory duration (white triangle). Biv: in addition, stimuli at more caudal locations elicited expiratory lengthening after stimulus offset consistent with Breuer–Hering expiratory reflex (gray square). Bv: suppression of inspiration throughout the duration of the stimulus (inspiratory brake, black square). Bvi: finally, in regions caudal and dorsal to the pre-Bötzinger complex, stimuli evoked inspiratory bursts, enabling entrainment of respiratory rhythm to a frequency slightly faster than the free-running behavior (black circle). I brake, inspiratory brake.