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. 2016 Apr;224:62-70.
doi: 10.1016/j.resp.2015.08.007. Epub 2015 Aug 24.

Studying Respiratory Rhythm Generation in a Developing Bird: Hatching a New Experimental Model Using the Classic in Vitro Brainstem-Spinal Cord Preparation

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Studying Respiratory Rhythm Generation in a Developing Bird: Hatching a New Experimental Model Using the Classic in Vitro Brainstem-Spinal Cord Preparation

Michael A Vincen-Brown et al. Respir Physiol Neurobiol. .
Free PMC article

Abstract

It has been more than thirty years since the in vitro brainstem-spinal cord preparation was first presented as a method to study automatic breathing behaviors in the neonatal rat. This straightforward preparation has led to an incredible burst of information about the location and coordination of several spontaneously active microcircuits that form the ventrolateral respiratory network of the brainstem. Despite these advances, our knowledge of the mechanisms that regulate central breathing behaviors is still incomplete. Investigations into the nature of spontaneous breathing rhythmicity have almost exclusively focused on mammals, and there is a need for comparative experimental models to evaluate several unresolved issues from a different perspective. With this in mind, we sought to develop a new avian in vitro model with the long term goal to better understand questions associated with the ontogeny of respiratory rhythm generation, neuroplasticity, and whether multiple, independent oscillators drive the major phases of breathing. The fact that birds develop in ovo provides unparalleled access to central neuronal networks throughout the prenatal period - from embryo to hatchling - that are free from confounding interactions with mother. Previous studies using in vitro avian models have been strictly limited to the early embryonic period. Consequently, the details and even the presence of brainstem derived breathing-related rhythmogenesis in birds have never been described. In the present study, we used the altricial zebra finch (Taeniopygia guttata) and show robust spontaneous motor outflow through cranial motor nerve IX, which is first detectable on embryonic day four and continues through prenatal and early postnatal development without interruption. We also show that brainstem oscillations change dramatically over the course of prenatal development, sometimes within hours, which suggests rapid maturational modifications in growth and connectivity. We propose that this experimental preparation will be useful for a variety of studies aimed at testing the biophysical and synaptic properties of neurons that participate in the unique spatiotemporal patterns of avian breathing behaviors, especially in the context of early development.

Keywords: Birds; Breathing; Central pattern generation; Development and maturation; In vitro brainstem-spinal cord preparation; Zebra finch.

Figures

Figure 1
Figure 1
Anatomy of the zebra finch brainstem showing the relative positions of spinal (C1 & 2) and cranial (V–XII) nerve roots. The electrophysiological recording site is marked by * in both panels. A: Ventral view of archetypal mature avian brainstem and cervical spinal cord from the pontomedullary border rostrally to the spinomedullary border caudally. These are the boundaries of the experiments described in the text. P=pons; M=medulla; V1=ophthalmic branch of V; V2=maxillary branch of V; V3=mandibular branch of V. Drawing modified from Bubien-Waluszewska, 1981. B: Ventral/ventrolateral view of the in vitro brainstem spinal cord en bloc plus recording chamber with suction electrode targeting cranial nerve IX shown with the broken white line outline. Cranial nerve XI is shown transected near spinomedullary border. Inset depicts a section of the rectified and integrated cranial nerve IX signal from this embryo. CN=cranial nerve.
Figure 2
Figure 2
Developmental changes in spontaneous neural activity and respiratory-related activity in the zebra finch brainstem from embryonic day 4 (E4) through external pipping or hatching (E14). A–K: Representative examples of rectified and integrated suction electrode measurements positioned on the transected stump of cranial nerve IX in vitro for each day of incubation. Spontaneous neural activity was not detected prior to E4. Short duration episodes were defined as episodes ≤ 25 s (see panels A. and G.) and long duration episodes were defined as episodes ≥ 40 s (see panels D. and G.).
Figure 3
Figure 3
Summary data for cranial nerve IX episode characteristics during embryonic development. A: Mean frequency of short duration (SD) and long duration (LD) episodes from E4–14. B: Mean duration for SD and LD episodes from E4–E14. Black bars=SD episodes and white bars=LD episode. Values are means ± standard error of the mean (SEM).
Figure 4
Figure 4
Distribution of in vitro brainstem spinal cord preparations that contained short duration (SD) and long duration (LD) episodes relative to total number preparations studied from each embryonic day (E4–14). Black bars=SD episodes and white bars=LD episodes.
Figure 5
Figure 5
Effects of ionotropic glutamate antagonism on short duration cranial nerve IX episode characteristics during embryonic development. A: Mean frequency of short duration (SD) episodes as percent of control frequency following bath application of CNQX (2–20 µM) and AP5 (50 µM) from E4–14. B: Mean duration of short duration (SD) episodes as percent of control frequency following bath application of CNQX (2–20 µM) and AP5 (50 µM) from E4–14. Black bars=SD episodes. Values are means ± standard error of the mean (SEM). * indicates P < 0.05.
Figure 6
Figure 6
Effects of ionotropic glutamate antagonism on long duration cranial nerve IX episode characteristics during embryonic development. A: Mean frequency of long duration (LD) episodes as percent of control frequency following bath application of CNQX (2–20 µM) and AP5 (50 µM) from E7–14. B: Mean duration of long duration (LD) episodes as percent of control frequency following bath application of CNQX (2–20 µM) and AP5 (50 µM) from E7–14. White bars=LD episodes. Values are means ± standard error of the mean (SEM). * indicates P < 0.05.
Figure 7
Figure 7
Effects of ionotropic and metabotropic glutamate receptor antagonism on rhythmic cranial IX nerve activity at E11–12. A: Example rectified integrated suction electrode recording showing the effect of ionotropic glutamate block (CNQX, 10 µM and AP5, 50 µM) on composite SD-LD episodes in an E11.5 embryo. Note the inhibition of SD episodes but not LD episodes. B: Example rectified integrated suction electrode recording showing the effect of metabotropic glutamate block, including 50 µM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) to block mGlu1 receptors, 100 µM 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) to block mGlu5 receptors, and 100 µM RS)-1-amino-5-phosphonoindan-1-carboxylic acid (RS)-APICA to block mGlu2/3 receptors, on composite SD-LD episodes in an E12 embryo. Note the inhibition of LD episodes but not SD episodes.

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