Maturation of Breathing-Related Inhibitory Neurotransmission in the Medulla Oblongata of the Embryonic and Perinatal Zebra Finch (Taeniopygia guttata).

The medullary portion of the embryonic zebra finch hindbrain was isolated and superfused with physiologically relevant artificial cerebral spinal fluid. This in vitro preparation produced uninterrupted rhythmic episodes of neural activity via cranial nerve IX (glossopharyngeal) from embryonic day 4 (E4) through hatching on E14. Cranial nerve IX carries motor activity to the glottis during the inspiratory phase of breathing, and we focused on the role of synaptic inhibition during the embryonic and perinatal maturation of this branchiomotor outflow. We show that spontaneous neural activity (SNA) is first observed on E4 and temporally transforms as the embryo ages. To start, SNA is dependent on the excitatory actions of GABAA and glycine. As the embryo continues to develop, GABAergic and glycinergic neurotransmission take on a modulatory role, albeit an excitatory one, through E10. After that, data show that GABAergic and glycinergic neurotransmission switches to a phenotype consistent with inhibition, coincident with the onset of functional breathing. We also report that the inhibitory action of GABAergic and glycinergic receptor gating is not necessary for the spontaneous generation of branchiomotor motor rhythms in these birds near hatching. This is the first report focusing on the development of central breathing-related inhibitory neurotransmission in birds during the entire period of embryogenesis.

Activity-dependent plasticity in the isolated embryonic avian brainstem following manipulations of rhythmic spontaneous neural activity.

When rhythmic spontaneous neural activity (rSNA) first appears in the embryonic chick brainstem and cranial nerve motor axons it is principally driven by nicotinic neurotransmission (NT). At this early age, the nicotinic acetylcholine receptor (nAChR) agonist nicotine is known to critically disrupt rSNA at low concentrations (0.1-0.5µM), which are levels that mimic the blood plasma levels of a fetus following maternal cigarette smoking. Thus, we quantified the effect of persistent exposure to exogenous nicotine on rSNA using an in vitro developmental model. We found that rSNA was eliminated by continuous bath application of exogenous nicotine, but rSNA recovered activity within 6-12h despite the persistent activation and desensitization of nAChRs. During the recovery period rSNA was critically driven by chloride-mediated membrane depolarization instead of nicotinic NT. To test whether this observed compensation was unique to the antagonism of nicotinic NT or whether the loss of spiking behavior also played a role, we eliminated rSNA by lowering overall excitatory drive with a low [K(+)]o superfusate. In this context, rSNA again recovered, although the recovery time was much quicker, and exhibited a lower frequency, higher duration, and an increase in the number of bursts per episode when compared to control embryos. Importantly, we show that the main compensatory response to lower overall excitatory drive, similar to nicotinergic block, is a result of potentiated chloride mediated membrane depolarization. These results support increasing evidence that early neural circuits sense spiking behavior to maintain primordial bioelectric rhythms. Understanding the nature of developmental plasticity in the nervous system, especially versions that preserve rhythmic behaviors following clinically meaningful environmental stimuli, both normal and pathological, will require similar studies to determine the consequences of feedback compensation at more mature chronological ages.

Studying respiratory rhythm generation in a developing bird: Hatching a new experimental model using the classic in vitro brainstem-spinal cord preparation.

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.