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

https://doi.org/10.1016/j.resp.2014.11.004Get rights and content

Highlights

  • Respiratory neurons were identified in adult turtle brainstems.

  • Ten percent of respiratory neurons were intrinsically bursting neurons.

  • Synaptic inhibition blockade resulted in seizure-like activity.

  • Intrinsically bursting neurons may contribute to turtle breathing.

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.

Introduction

Neurons with intrinsic bursting properties spontaneously fire bursts of action potentials in the absence of synaptic inputs. Early analysis of invertebrate neural networks suggested that intrinsically bursting neurons were abundant in networks that continuously produced rhythmic motor behavior (Getting, 1988). Since breathing is a motor behavior that is required from birth to death, a subpopulation of interconnected respiratory intrinsically bursting neurons was postulated to contribute to respiratory rhythm generation (Feldman and Cleland, 1982). Consistent with this hypothesis, intrinsically bursting respiratory neurons are found in perinatal rodent in vitro preparations that produce respiratory-related motor output, especially in the pre-Bötzinger Complex (preBötC) (Smith et al., 1991, Johnson et al., 1994, Rekling and Feldman, 1998, Thoby-Brisson and Ramirez, 2000, Thoby-Brisson and Ramirez, 2001, Peña et al., 2004) and para-Facial Respiratory Group (pFRG; Onimaru et al., 1989, Onimaru et al., 1995, Onimaru and Homma, 2003). The preBötC and the pFRG are coupled oscillatory networks that are hypothesized to be the sites of inspiratory and expiratory rhythm generation (Feldman and Del Negro, 2006, Feldman et al., 2013). The hybrid pacemaker-network model proposed that intrinsically bursting neurons played a critical role in respiratory rhythm generation (Funk and Feldman, 1995, Ramirez et al., 1997, Butera et al., 1999a, Butera et al., 1999b, Koshiya and Smith, 1999, Smith et al., 2000, Peña et al., 2004). This hypothesis, however, is criticized because rhythmic activity persists when specific ion currents underlying intrinsic bursting activity are blocked (Del Negro et al., 2005). Instead, respiratory rhythm generation is hypothesized to require excitatory synaptic transmission in the dendrites to activate inward currents to produce the large depolarizing burst in respiratory neurons (“group pacemaker” model; Rekling and Feldman, 1998, Del Negro et al., 2002, Feldman and Del Negro, 2006, Mironov, 2008, Rubin et al., 2009, Del Negro et al., 2010, Feldman et al., 2013).

Intrinsically bursting respiratory neurons are hypothesized to be expressed primarily in young mammals, and sparsely expressed in adult mammals as synaptic inhibition increases and becomes the dominant mechanism for rhythm generation (Richter and Spyer, 2001, Broch et al., 2002, Richter and Smith, 2014). However, this hypothesis has not been tested in fully mature mammals, and it is not known whether intrinsically bursting neurons are expressed or contribute to respiratory rhythm generation in fully mature rodents, other mammals, or in non-mammalian vertebrates, in part due to significant technical difficulties in the experimental approach. At best, intrinsically bursting respiratory neurons are found in the preBötC of older perfused juvenile rat (P14–P21) and mouse (P21–P42) preparations (Paton, 1997, St-John et al., 2009). Second, most studies examining intrinsically bursting respiratory neurons focused on the preBötC and pFRG regions and did not test neurons in other brainstem regions. Third, no studies have tested for intrinsically bursting respiratory neurons in ectothermic vertebrates and thereby added a comparative and evolutionary perspective to this debate.

For ectothermic vertebrates, the potential existence of intrinsically bursting respiratory neurons was suggested by showing that rhythmic activity persists during synaptic inhibition blockade in isolated brainstems from tadpoles (Galante et al., 1996, Broch et al., 2002), adult lampreys (Rovainen, 1983), and adult turtles (Johnson et al., 2002). With synaptic inhibition blocked or severely attenuated, the persistent rhythm is thought to be due to intrinsically bursting respiratory neurons that continue to generate a wave of excitatory synaptic drive through the respiratory network to the respiratory motoneurons. Although this is the working hypothesis, experimental evidence directly demonstrating this principle is lacking. Finally, no studies have tested whether respiratory neurons in ectothermic vertebrates have intrinsic bursting properties.

To address these questions, brainstems from adult red-eared slider turtles were isolated under in vitro conditions and silicon multichannel electrodes were used to identify respiratory neurons and test for intrinsic bursting properties. Isolated turtle brainstems are advantageous because they produce expiratory- and inspiratory-related motor output that is qualitatively similar to that produced by intact turtles (Johnson and Mitchell, 1998). Also, since this turtle species is extremely resistant to hypoxia (Jackson, 2000, Johnson et al., 1998), respiratory motor output can be produced on the XII nerve root of isolated turtle brainstems for several days at physiologically relevant temperatures (Wilkerson et al., 2003). Our goals were to: (1) identify respiratory neurons in the turtle brainstem and expand on the initial map generated by Takeda et al. (1986); (2) classify respiratory neurons based on their firing pattern relative to the XII motor discharge; (3) test whether respiratory neurons express intrinsic bursting properties by blocking synaptic transmission (via a cocktail of drugs that block excitatory and inhibitory synaptic transmission); (4) determine whether intrinsic bursting respiratory neurons belong to a specific type or are localized within specific brainstem region; and (5) test whether synaptic inhibition blockade transforms the firing pattern of respiratory neurons or results in non-specific seizure-like activity. Preliminary reports of this work were published in abstract form (Chapman and Johnson, 2008).

Section snippets

Methods

All procedures were approved by the Animal Care and Use Committee at the University of Wisconsin-Madison School of Veterinary Medicine. Adult red-eared slider turtles (Trachemys scripta, n = 21, 689 ± 200 g) were obtained from commercial suppliers and kept in a large open tank where they had access to water for swimming and heat lamps and dry areas for basking. Room temperature was set to 27–28 °C with light provided 14 h/day. Turtles were fed ReptoMin® floating food sticks (Tetra, Blackburg, VA, USA)

Respiratory neuron classification and location

While recording respiratory motor output on XII nerves in isolated adult turtle brainstems (n = 15), silicon multichannel recording electrodes were placed in the brainstem lateral to the midline on either side. Respiratory (n = 219) and non-respiratory neurons (n = 6) were recorded in the brainstem in an area extending from caudal to the XII nerve root to caudal to the vestibulocochlear (VIII) and abducens (VI) nerve roots (Table 1; Fig. 3). Expiratory “E-cells” fired action potentials almost

Discussion

The main findings were that ∼10% of respiratory neurons in isolated turtle brainstems have intrinsic bursting properties. These neurons were distributed within several different classes of respiratory neurons with post-inspiratory neurons having the highest percentage of neurons expressing these properties. Most of the intrinsically bursting respiratory neurons were clustered at the level of the X nerve root. This study also expanded the scope of previously recorded respiratory neurons in

Acknowledgements

This work was supported by the National Science Foundation (IOB 0517302 to SMJ), American Physiological Society (Research Career Enhancement Award to MSH), and National Institutes of Health (NIH T32 GM007507 to BMK).

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