Respiratory neuron characterization reveals intrinsic bursting properties in isolated adult turtle brainstems (Trachemys scripta)

Abstract

It is not known whether respiratory neurons with intrinsic bursting properties exist within ectothermic vertebrate respiratory control systems. Thus, isolated adult turtle brainstems spontaneously producing respiratory motor output were used to identify and classify respiratory neurons based on their firing pattern relative to hypoglossal (XII) nerve activity. Most respiratory neurons (183/212) had peak activity during the expiratory phase, while inspiratory, post-inspiratory, and novel pre-expiratory neurons were less common. During synaptic blockade conditions, ∼10% of respiratory neurons fired bursts of action potentials, with post-inspiratory cells (6/9) having the highest percentage of intrinsic burst properties. Most intrinsically bursting respiratory neurons were clustered at the level of the vagus (X) nerve root. Synaptic inhibition blockade caused seizure-like activity throughout the turtle brainstem, which shows that the turtle respiratory control system is not transformed into a network driven by intrinsically bursting respiratory neurons. We hypothesize that intrinsically bursting respiratory neurons are evolutionarily conserved and represent a potential rhythmogenic mechanism contributing to respiration in adult turtles.

Keywords: Chelonian; Pacemaker; Reptile; Rhythm generation.

Fig. 1

Multichannel recording in isolated turtle brainstem producing spontaneous respiratory motor output. (A) Drawing of turtle brainstem in vitro with suction electrode attached to XII nerve root and two silicon multichannel electrodes inserted into the brainstem. (B) Magnification of a single silicon multichannel electrode with four shanks (black circles on shanks represent recording sites). (C) Spontaneous respiratory motor output recorded on XII nerve root. (D, E) Integrated XII motor output and extracellular recordings from respiratory-related neurons under control conditions (aCSF solution; left panels) and during synaptic transmission blockade (right panels). Multiple neurons were recorded in aCSF solution and one neuron in each case exhibited intrinsic bursting properties during synaptic transmission blockade (right panels).

Fig. 2

Cycle-triggered histograms are shown for two types of expiratory neurons, such as an E-cell (A) and EI-cell (B); an inspiratory neuron, such as an I-cell (C); two types of post-inspiratory cells, such as a Post-I-cell (D) and an E-cell/Post-I-cell; and a pre-expiratory cell, such as a Pre-E-cell (F). The light gray line represents the averaged XII respiratory motor burst with the average action potential frequency graphed in black histograms (0.5 s bins).

Fig. 3

Diagrams of turtle hemibrainstems with approximate locations of specific nuclei (labeled light gray shapes) included for reference, such as the abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), and nucleus ambiguus (Amb), which were modified from Cruce and Nieuwenhuys (1974). The location of most neurons within each class is shown. The position of the neurons were not located with respect to depth and mediolateral location. Open circles indicate neuron location, while filled circles indicate that the neuron had intrinsic bursting properties.

Fig. 4

Examples of respiratory neurons in aCSF solution (left panels) that have intrinsic bursting properties when bathed in synaptic blockade solution containing CNQX, APV, strychnine, and bicuculline with (right panels). The intrinsically bursting respiratory neurons include an E-cell (A), EI-cell (B), E-cell/Post-I-cell (C), and a Pre-E-cell (D). The integrated respiratory motor output recorded on XII nerve root is shown as a blue trace and the timing of action potential firing is indicated by the vertical lines. (E) Diagrams of turtle hemibrainstem with the rostrocaudal location of all intrinsically bursting respiratory neurons shown (open circles).

Fig. 5

Seizure activity in turtle brainstem induced by synaptic inhibition blockade. (A) Recordings of integrated motor output on roots of cranial nerves V, IX, and XII under control conditions (aCSF solution; left panels), and during synaptic inhibition blockade (picrotoxin, strychnine; right panels). Rhythmic activity on IX and XII roots was transformed into large synchronous bursts on all roots. (B) Recordings of integrated XII motor output along with 16 extracellular recordings from a pontine site rostral to cranial nerve root VI (see diagram at right) under control conditions (left panel) and during synaptic inhibition blockade (right panel). Synchronized bursts of action potentials were observed on all channels during synaptic inhibition blockade.