Glycinergic inhibition is essential for co-ordinating cranial and spinal respiratory motor outputs in the neonatal rat

Abstract

Eupnoeic breathing in mammals is dependent on the co-ordinated activity of cranial and spinal motor outputs to both ventilate the lungs and adjust respiratory airflow, which they do by regulating upper-airway resistance. We investigated the role of central glycinergic inhibition in the co-ordination of cranial and spinal respiratory motor outflows. We developed an arterially perfused neonatal rat preparation (postnatal age 0-4 days) to assess the effects of blocking glycine receptors with systemically administered strychnine (0.5-1 microM). We recorded respiratory neurones located within the ventrolateral medulla, inspiratory phrenic nerve activity (PNA) and recurrent laryngeal nerve activity (RLNA), as well as dynamic changes in laryngeal resistance. Central recordings of postinspiratory neurones revealed an earlier onset in firing relative to the onset of inspiratory PNA after exposure to strychnine (260 +/- 38.9 vs. 129 +/- 26.8 ms). After glycine receptor blockade, postinspiratory neurones discharged during the inspiratory phase. Strychnine also evoked a decrease in PNA frequency (from 38.6 +/- 4.7 to 30.7 +/- 2.8 bursts min(-1)), but amplitude was unaffected. In control conditions, RLNA comprised inspiratory and postinspiratory discharges; the amplitude of the latter exceeded that of the former. However, after administration of strychnine, the amplitude of inspiratory-related discharge increased (+65.2 +/- 15.2 %) and exceeded postinspiratory activity. Functionally this change in RLNA caused a paradoxical, inspiratory-related glottal constriction during PNA. We conclude that during the first days of life in the rat, glycine receptors are essential for the formation of the eupnoeic-like breathing pattern as defined by the co-ordinated activity of cranial and spinal motor inspiratory and postinspiratory activities.

Figure 1. Two types of postinspiratory neurones found in arterially perfused neonatal rats

An illustration of the heterogeneity of postinspiratory neurones recorded intracellularly in the ventral respiratory group of neonatal rats. A, type-1 postinspiratory neurones were characterised by an inspiratory-related hyperpolarisation with rebound depolarisation and discharge (arrows) that slowly declined in frequency during the expiratory interval. B, type-2 postinspiratory neurones also exhibited inspiratory-related hyperpolarisation, but this was followed by a brief, high-frequency volley of action potentials that was restricted to early expiration. C, histogram of the discharge duration of all recorded postinspiratory neurones illustrating two separate modes. Please note that the twisted curves of the Gaussian distribution are due to logarithmic scaling of the x-axis. n, number of neurones; P, postnatal age; PNA, phrenic nerve activity; WHBP, working heart-brainstem preparation.

Figure 2. Strychnine application induces firing of postinspiratory neurones during inspiration

Two types of postinspiratory neurones (type-1 (A) and type-2 (B)) found in arterially perfused neonatal rats. A and B, the inspiratory-related hyperpolarisation of a type-1 and type-2 postinspiratory neurone was reduced and converted into depolarisation after application of strychnine (A, 0.5 μM; B, 1 μM). Consequently these neurones fired during the inspiratory phase of PNA. C, example of a type-2 postinspiratory neurone recorded extracellularly that lost its inspiratory modulation and exerted tonic discharge throughout the respiratory cycle after application of strychnine (1 μM). Note that preinspiratory inhibition was revealed in type-1 neurones (n = 3), which tended to persist in type-2 neurones (n = 4, see arrows in A and B). P, postnatal age.

Figure 3. Blockade of glycine receptors depresses postinspiratory activity but augments inspiratory discharge in the recurrent laryngeal nerve

A, in the recurrent laryngeal nerve from a neonatal rat that was a few hours old there were both inspiratory-related (i.e. coincident with PNA) and postinspiratory discharges. Note that postinspiratory activity is much greater in amplitude than inspiratory motor discharge in control conditions. B, the influence of systemic application of strychnine (0.5 μM) on PNA and recurrent laryngeal nerve activity (RLNA) is shown. Note the dramatic increase in the amplitude of the inspiratory-related discharge in the RLNA after glycine receptor antagonism. This was accompanied by a reduction in postinspiratory activity. *Expanded view of one cycle illustrating the respiratory phases (I, inspiration; PI, postinspiration; E, expiration) before and after application of strychnine. Note the almost complete absence of postinspiratory activity after glycine receptor blockade. P, postnatal age.

Figure 4. Duration of respiratory phases in RLNA is correlated with the frequency of phrenic nerve bursts

Diagram illustrating the linear correlation of the duration of the individual respiratory phases (▵ inspiration, ○ postinspiration and ▪ expiration) versus the respiratory cycle length calculated from the interval between two inspiratory bursts of PNA under control conditions. Note that the prolongation of each respiratory phase with increasing cycle length was correlated (postinspiration r = 0.870, degrees of freedom, d.f. 13, P < 0.001; expiration r = 0.856, d.f. 13, P < 0.001; inspiration r = 0.532, d.f., 13, P < 0.05). CL, respiratory cycle length.

Figure 5. Respiratory frequency and pattern changes induced by glycine receptor blockade in neonatal rats

A, bar graph illustrating the effect of strychnine on the integrals of integrated inspiratory and postinspiratory activities recorded from the recurrent laryngeal nerve (n = 9; contr., control; stry., strychnine). B, bar graph illustrating the influence of 0.5-1 μM strychnine on the duration of the three respiratory phases (inspiration, postinspiration and expiration) as measured from recordings of RNLA. Please note that the brackets over integrated inspiratory and postinspiratory activity reflect a significant difference in the discharge pattern (from PI exceeding I to I exceeding PI discharge) revealed by ANOVA with interaction. *P < 0.05, **P < 0.01. CL, cycle length; E, expiration; I, inspiration; PI, post-inspiration.

Figure 6. Strychnine shifts glottic constriction into inspiration

A, the dynamic changes in glottal resistance during the respiratory cycle included dilatation during inspiration (decrease in sub-glottal pressure or SGP) and constriction in postinspiration (shaded area; increase in SGP) in a 3-day-old rat (P3). B, after strychnine blockade of glycine receptors glottal constriction occurred earlier during neural inspiration. C, bar diagram comparing the integral of integrated PNA that occurred from the onset to the peak rise in SGP (shaded areas in A and B), which was analysed in control conditions and after exposure to strychnine (n = 8). **P < 0.01.

Figure 7. Effect of a high dose of strychnine (> 2 μM) on PNA and upper-airway resistance

A, the eupnoeic modulation of laryngeal resistance as revealed by changes in SGP (see Fig. 6). B, application of strychnine increased the frequency and duration of inspiratory PNA. These periods of fast and tonic PNA were accompanied by periods of persistent increases in SGP on which was superimposed further laryngeal constrictions that were coincident with PNA bursting. The latter indicates paradoxical glottic constriction during neural inspiration. P, postnatal age.