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, 78 (11), 1081-1096

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

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

Kaci L Pickett et al. Dev Neurobiol.

Abstract

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.

Keywords: GABA; birds; breathing; development; embryo; glycine; hindbrain; inhibitory.

Figures

Figure 1.
Figure 1.
Rhythmic cranial nerve IX activity produced by the zebra finch medulla in vitro from embryonic day 4 (E4) through external pipping (E14). Each day of incubation includes a representative example of a rectified, integrated and averaged suction electrode measurement in the presence of 3 mM K+ and 1.26 mM Ca2+ aCSF (left panel). The right panels shows a representative episode for each day between E4 and E14 with an expanded time base.
Figure 2.
Figure 2.
Summary data of glossopharyngeal nerve (cranial nerve IX) activity during developmental days E4-E14. (A.) Mean episode frequency (min−1); (B.) Mean episode duration (sec); (C.) Mean discharge frequency for developmental periods encompassing E4-6, E7-10 and E11-14; (D.) Mean duration for developmental periods encompassing E4-6, E7-10 and E11-14. Values are means ± SEM. Unique lowercase letters (a, b, c.) above the bars on individual embryonic days indicate statistical differences among ages with P<0.05.
Figure 3.
Figure 3.
Summary data of glossopharyngeal nerve (cranial nerve IX) activity following bath application of GABA/glycine antagonists from E4 through hatching (E14). (A.) Episode frequency as a percent of control; (B.) Episode duration as a percent of control; (C.) Episode frequency as a percent of control for developmental periods encompassing E4-6, E7-10 and E11-14; (D.) Episode duration as a percent of control for developmental periods encompassing E4-6, E7-10 and E11-14. Values are means ± SEM. * above bars in panels A. and B. indicates differences from control (P<0.05). For grouped means in panels C. (frequency) and D. (duration), unique letters above bars indicate differences in age groups (P<0.05) and + above bars indicates differences from control (P<0.05).
Figure 4.
Figure 4.
Example recordings of rectified and integrated cranial nerve IX activity before and during application of GABAA and glycine antagonists. (A.) Shows rhythmic episodes generated at E6 in control (left panel) and following bath application of bicuculline (5 μM) and strychnine (0.4 μM) (right panel); (B.) Shows rhythmic episodes generated at E8 in control (left panel) and following bath application of bicuculline (5 μM) and strychnine (0.4 μM) (right panel); (C.) Shows rhythmic episodes generated at E8 in control (left panel) and following bath application of bicuculline (5 μM) and strychnine (0.4 μM) (right panel). Each panel (horizontally) in A., B., and C. represents the same animal and experiment.
Figure 5.
Figure 5.
Summary data for the effects of GABAA agonist muscimol on rhythmic cranial nerve IX activity in the zebra finch with data grouped into embryonic ages E6-E10, and E12-E14. (A.) Shows rhythmic episodes generated at E7 in control and following bath application of muscimol (0.3 μM) (right panel); (B.) Shows rhythmic episodes generated at E12 in control and following bath application of muscimol (0.3 μM). Each panel (horizontally) in A. and B. represents the same animal and experiment. (C.) Shows mean episode frequency as a percent of control in each age group; (D.) Shows mean episode duration as a percent of control in each age group; Values in C. and D. are means ± SEM. Unique letters (a, b) indicate difference between ages P<0.05 and + indicates difference from control P<0.05.
Figure 6.
Figure 6.
Example recordings of rectified and integrated cranial nerve IX activity and summary data comparing rhythmic branchiomotor features from the zebra finch medullary and the thick medullary slice preparation. In all cases, bars represent mean cranial nerve IX activity with data grouped into embryonic ages E6-E10, and E12-E14. (A.) Inset depicts the approximate boundaries of the medullary slice: sn1=spinal nerve 1; cn12=cranial nerve 12; cn9/10=cranial nerve 9/10; cn7/8=cranial nerve 7/8; cn5=cranial nerve 5. The left panel (A.) shows rhythmic episodes generated at E8 in control conditions using the entire medulla and then following the transection of the medulla 500 μM above and below cranial nerve IX (right panel); (B.) The left panel shows rhythmic episodes generated at E12 in control conditions using the entire medulla and then following the transection of the medulla 500 μM above and below cranial nerve IX (right panel); (C.) Episode frequency as a percent of control (i.e., entire medulla) following transections that produce a 1000-micron thick slice of the medulla; (D.) Episode duration as a percent of control (i.e., entire medulla) following transections that produce a 1000-micron thick slice of the medulla. Values are means ± SEM. + indicates difference from control P<0.05.
Figure. 7.
Figure. 7.
Example recordings of rectified and integrated cranial nerve IX activity and summary data examining the effects of bicuculline and strychnine antagonism on branchiomotor output using the thick medullary slice. (A.) Shows cranial nerve IX activity during baseline conditions at E8 and then following bath application of bicuculline (5 μM) and strychnine (0.4 μM), which was followed by a washout period. (B.) Shows cranial nerve IX activity during baseline conditions at E12 and then following bath application of bicuculline (5 μM) and strychnine (0.4 μM), which was followed by a washout period. Each panel (horizontally) represents the same animal and experiment. (C.) Shows mean episode frequency as a percent of control following bath application of GABA/glycine antagonists in the thick slice preparation for ages E6-10 and E12-14; (D.) Shows episode duration as a percent of control following bath application of GABA/glycine antagonists in the thick slice preparation for ages E6-10 and E12-14. Values are means ± SEM. Unique letters (a, b, c) indicate difference between ages P<0.05 and + indicates difference from control P<0.05.

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