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.

The developing brainstem generates periodic spontaneous neural activity (SNA). SNA is thought to be crucial for the maturation of motor neurons and associated neural networks. Beginning around embryonic day five (E5), SNA is driven by cholinergic nicotinic neurotransmission (NT) with only minor modulatory inputs from ƴ‐aminobutyric acid GABA/glycinergic NT. Here we tested the effects of embryonic nicotine exposure on the neurogenesis of SNA in brainstem regions that later support homeostatic functions such as breathing. We hypothesized that brain stem motor circuits will compensate for nicotine blockade of nicotine acetylcholine receptors (nAChR) to maintain SNA by rapidly shifting to GABA/glycinergic NT. To test our hypothesis we used electrical physiological recordings and pharmacology before and after chronic nicotine exposure to probe for nicotine evoked plasticity of brain stem derived SNA in chick embryos on E5‐E5.5. Results show that nicotine abolished SNA at millimolar concentrations similar to those detected in human plasma following a single cigarette. However SNA recovered quickly (蠄 4 h) while still in the presence of nicotine suggesting that compensatory neurotransmitter(s) was driving SNA activity. Indeed, results show that GABA/glycinergic NT is required to drive SNA in the absence of nAChR availability demonstrating a form of activity dependent neural plasticity. In summary, our results show that the developing brain stem can rapidly adapt to perturbations of previously well‐defined CNS neurotransmitters to maintain SNA, and suggests that neurotransmitter phenotypic plasticity is a critical feature of the embryonic brainstem that may function to maintain the integrity of homeostatic oscillatory network functions in vivo.

Brain‐related breathing problems are common in early life. Despite intensive investigations into mammalian breathing control, our understanding of respiratory rhythm generation is still unclear, especially during the prenatal period. We aim to develop the avian embryo as a more tractable prenatal model to study breathing related behaviors. Birds are highly active endotherms, like mammals, and provide access for manipulations that mammals cannot. In the rodent, automatic breathing behaviors have been identified in isolated transverse brainstem slices. We hypothesize that there is a similar complex within the avian brainstem. Thus, our goal is to identify the central pattern generator for breathing in the bird. We used an activity dependent dye, electrical recordings, and Nissl staining to generate a neuroanatomical brain stem map. We examined isolated brain stems from Corturnix japonica near internal pipping when breathing movements are present. Results suggest that birds possess a spontaneously generated rhythm in the ventral lateral medulla similar to mammals. We further show that in some preparations tonic activity became rhythmic when inhibitory neurotransmission was blocked. These results suggest critical differences between the prenatal development of central breathing control of bird and mammals, including the importance of GABA and glycine neurotransmission prior to parturition.

Activity in the CNS begins as a widely distributed and synchronous depolarization wave known as rhythmic spontaneous neural activity (rSNA). As growth and maturation proceed, rSNA differentiates into central pattern generators (CPGs) that produce individual rhythmic behaviors, such as breathing. While it is important to understand the mechanisms that underlie this progression, monitoring the uninterrupted development of breathing‐related motor circuits remains difficult due to the limited access of these circuits in the mammalian model and a lack of information about breathing‐related CPG behavior in other models, such as birds. Here, we use a novel brainstem spinal cord preparation from the altricial Zebra Finch embryo to study the early development and maturation of breathing‐related motor patterns conveyed in cranial nerve IX. We show that rSNA can be measured continuously from embryonic day 4 (E4) through external pipping (E14). rSNA starts at embryonic day 4 (E4) with low frequency (0.29 bursts/min) short duration (<2.5 sec) single bursts, and progresses to a more complex pattern consisting of episodes with short duration bursts coupled to slow frequency (0.2 burst/minute) long duration bursts (>80 sec) at E9. The activity returns to short duration (<2.5 sec), high frequency (6‐12 bursts/min) at hatching (E14). At E4 the activity is nicotinergic‐sensitive and in the older animals (>E7) the short duration bursts are completely blocked by NMDA and non‐NMDA antagonism. The long duration bursts only diminished by bath application of m‐type glutamate antagonists. These findings establish an extremely tractable new experimental model to study the development of central breathing behaviors in both health and disease.