Vitaliy Marchenko

Different patterns of respiratory and cardiovascular responses elicited by chemical stimulation of dorsal medulla in the rat

Respiratory and cardiovascular responses to microinjections (10 nl) of L-glutamate (10 mM) into the dorsal medulla were studied in spontaneously breathing urethane-anesthetized, adult male Wistar rats. A total of 10 patterns of respiratory and cardiovascular responses were observed: (1) hypotension alone; (2) hypotension and bradycardia; (3) hypotension and apnea; (4) hypotension, bradycardia, and apnea; (5) apnea alone; (6) hypotension and fast and shallow breathing; (7) hypotension, bradycardia, and fast and shallow breathing; (8) fast and shallow breathing alone; (9) sighs; and (10) increase in BP and HR accompanied with fast and shallow breathing. The sites from which a combination of hypotension, bradycardia, and apnea was elicited, occupied a region in the medial subnucleus of nucleus tractus solitarius (nTS), the reticular formation just ventral to it, and the dorsal motor nucleus of vagus. The sites from which hypotension alone or a combination of hypotension and apnea were elicited occupied the margins of the medial subnucleus of nTS. The sites from which apnea alone was elicited were located in the ventrolateral part of nTS and the reticular formation just ventral to it. In the commissural subnucleus of nTS, the responses comparable to those elicited by peripheral chemoreceptor stimulation (i.e., increase in BP, HR, and respiratory rate) were located in a midline region just caudal to the calamus scriptorius, the sites from which sighs were elicited were located slightly lateral and deeper, the sites from which fast and shallow breathing were elicited were located in the dorsal portion, slightly lateral to the midline. These results are expected to prove useful in studies in which microinjection technique is used to identify transmitters/receptors involved in mediating respiratory and cardiovascular reflex responses.

Cardiovascular responses to chemical stimulation of the lateral tegmental field and adjacent medullary reticular formation in the rat

Relatively few studies have been done to characterize cardiovascular responses to the chemical stimulation of sites located in the medullary lateral tegmental field (LTF) and most of them have been carried out in anesthetized animals. Our experiments were carried out in decerebrated, artificially ventilated, adult male Wistar rats. In the LTF, two types of cardiovascular responses were elicited. One type consisted of pressor responses accompanied by bradycardia. Such responses were elicited from a region 0.4 mm caudal to 0.8 mm rostral to the calamus scriptorius (CS); maximum responses were elicited from a site 0.6 mm rostral to the CS, 1.2 mm lateral to the midline and 1.2 mm deep from the dorsal medullary surface. Another type consisted of pressor responses without any change in heart rate; such responses were elicited from a region 1-1.6 mm rostral to the CS. Nucleus ambiguus (nAmb) and dorsal motor nucleus of the vagus (nDMX) and the reticular formation surrounding these areas were the main sites from which bradycardia (accompanied by either no or small changes in BP) was elicited. In the nAmb, maximum bradycardia was elicited from a site 0.6 mm rostral to the CS, 1.8 mm lateral to the midline and 2.4 mm deep from the dorsal medullary surface. In the nDMX, most prominent bradycardic responses were elicited at 0-0.6 mm rostral to the CS, and 0.6 mm lateral to the midline and 1 mm deep from the dorsal medullary surface. Cardiovascular effects elicited from sites in other well-known areas, such as the rostral ventrolateral medullary pressor area (RVLM) and caudal ventrolateral medullary depressor area (CVLM), and the nucleus tractus solitarius (nTS) were also included for comparison of different responses. These results are expected to prove useful in studies in which the microinjection technique is used to characterize cardiovascular responses.

Perturbations of Respiratory Rhythm and Pattern by Disrupting Synaptic Inhibition within Pre-Bötzinger and Bötzinger Complexes

Visual Abstract The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. Current models postulate that both generation of the rhythm and coordination of the inspiratory-expiratory pattern involve inhibitory synaptic interactions within and between these regions. Both regions contain glycinergic and GABAergic neurons, and rhythmically active neurons in these regions receive appropriately coordinated phasic inhibition necessary for generation of the normal three-phase respiratory pattern. However, recent experiments attempting to disrupt glycinergic and GABAergic postsynaptic inhibition in the pre-BötC and BötC in adult rats in vivo have questioned the critical role of synaptic inhibition in these regions, as well as the importance of the BötC, which contradicts previous physiological and pharmacological studies. To further evaluate the roles of synaptic inhibition and the BötC, we bilaterally microinjected the GABAA receptor antagonist gabazine and glycinergic receptor antagonist strychnine into the pre-BötC or BötC in anesthetized adult rats in vivo and in perfused in situ brainstem–spinal cord preparations from juvenile rats. Muscimol was microinjected to suppress neuronal activity in the pre-BötC or BötC. In both preparations, disrupting inhibition within pre-BötC or BötC caused major site-specific perturbations of the rhythm and disrupted the three-phase motor pattern, in some experiments terminating rhythmic motor output. Suppressing BötC activity also potently disturbed the rhythm and motor pattern. We conclude that inhibitory circuit interactions within and between the pre-BötC and BötC critically regulate rhythmogenesis and are required for normal respiratory motor pattern generation.

Motoneuron firing patterns underlying fast oscillations in phrenic nerve discharge in the rat

Fast oscillations are ubiquitous throughout the mammalian central nervous system and are especially prominent in respiratory motor outputs, including the phrenic nerves (PhNs). Some investigators have argued for an epiphenomenological basis for PhN high-frequency oscillations because phrenic motoneurons (PhMNs) firing at these same frequencies have never been recorded, although their existence has never been tested systematically. Experiments were performed on 18 paralyzed, unanesthetized, decerebrate adult rats in which whole PhN and individual PhMN activity were recorded. A novel method for evaluating unit-nerve time-frequency coherence was applied to PhMN and PhN recordings. PhMNs were classified according to their maximal firing rate as high, medium, and low frequency, corresponding to the analogous bands in PhN spectra. For the first time, we report the existence of PhMNs firing at rates corresponding to high-frequency oscillations during eupneic motor output. The majority of PhMNs fired only during inspiration, but a small subpopulation possessed tonic activity throughout all phases of respiration. Significant time-varying PhMN-PhN coherence was observed for all PhMN classes. High-frequency, early-recruited units had significantly more consistent onset times than low-frequency, early/middle-recruited and medium-frequency, middle/late-recruited PhMNs. High- and medium-frequency PhMNs had significantly more consistent offset times than low-frequency units. This suggests that startup and termination of PhMNs with higher firing rates are more precisely controlled, which may contribute to the greater PhMN-PhN coherence at the beginning and end of inspiration. Our findings provide evidence that near-synchronous discharge of PhMNs firing at high rates may underlie fast oscillations in PhN discharge.

The role of spinal GABAergic circuits in the control of phrenic nerve motor output

While supraspinal mechanisms underlying respiratory pattern formation are well characterized, the contribution of spinal circuitry to the same remains poorly understood. In this study, we tested the hypothesis that intraspinal GABAergic circuits are involved in shaping phrenic motor output. To this end, we performed bilateral phrenic nerve recordings in anesthetized adult rats and observed neurogram changes in response to knocking down expression of both isoforms (65 and 67 kDa) of glutamate decarboxylase (GAD65/67) using microinjections of anti-GAD65/67 short-interference RNA (siRNA) in the phrenic nucleus. The number of GAD65/67-positive cells was drastically reduced on the side of siRNA microinjections, especially in the lateral aspects of Rexeds laminae VII and IX in the ventral horn of cervical segment C4, but not contralateral to microinjections. We hypothesize that intraspinal GABAergic control of phrenic output is primarily phasic, but also plays an important role in tonic regulation of phrenic discharge. Also, we identified respiration-modulated GABAergic interneurons (both inspiratory and expiratory) located slightly dorsal to the phrenic nucleus. Our data provide the first direct evidence for the existence of intraspinal GABAergic circuits contributing to the formation of phrenic output. The physiological role of local intraspinal inhibition, independent of descending direct bulbospinal control, is discussed.

GABAAergic and Glycinergic Inhibition in the Phrenic Nucleus Organizes and Couples Fast Oscillations in Motor Output

One of the characteristics of respiratory motor output is the presence of fast synchronous oscillations, at rates far exceeding the basic breathing rhythm, within a given functional population. However, the mechanisms responsible for organizing phrenic output into two dominant bands in vivo, medium (MFO)- and high (HFO)-frequency oscillations, have yet to be elucidated. We hypothesize that GABA(A)ergic and glycinergic inhibition within the phrenic motor nucleus underlies the specific organization of these oscillations. To test this, the phrenic nuclei (C(4)) of 14 unanesthetized, decerebrate adult male Sprague-Dawley rats were microinjected unilaterally with either 4 mM strychnine (n = 7) or GABAzine (n = 7) to block glycine or GABA(A) receptors, respectively. Application of GABAzine caused an increase in overall phrenic amplitude during all three phases of respiration (inspiration, postinspiration, and expiration), while the increases caused by strychnine were most pronounced during postinspiration. Neither antagonist produced changes in inspiratory duration or respiratory rate. Power spectral analysis of inspiratory phrenic bursts showed that blockade of inhibition caused significant reduction in the relative power of MFO (GABA(A) and glycine receptors) and HFO (GABA(A) receptors only). In addition, analysis of the coherence between the firing of the ipsi- and contralateral phrenic nerves revealed that HFO coupling was significantly reduced by both antagonists and that of MFO was significantly reduced only by strychnine. We conclude that both GABA(A) and glycine receptors play critical roles in the organization of fast oscillations into MFO and HFO bands in the phrenic nerve, as well as in their bilateral coupling.

Dynamic changes in phrenic motor output following high cervical hemisection in the decerebrate rat

Hemisection of the spinal cord at C2 eliminates ipsilateral descending drive to the phrenic nucleus and causes hemidiaphragmatic paralysis in rats. Phrenic nerve (PhN) or diaphragmatic activity ipsilateral to hemisection can occasionally be induced acutely following hemisection by respiratory stressors (i.e., hypercapnia, asphyxia, contralateral phrenicotomy) and becomes spontaneously active days-to-weeks later. These investigations, however, are potentially confounded by the use of anesthesia, which may suppress spontaneously-active crossed phrenic pathways. Experiments were performed on vecuronium-paralyzed, unanesthetized, decerebrate adult male rats and whole PhN activity recorded continuously before, during, and after high cervical hemisection at the C1 spinal level. Crossed phrenic activity recovered spontaneously over minutes-to-hours with maximal recovery of 11.8 ± 3.1% (m ± SE) in the PhN ipsilateral to hemisection. Additionally, there was a significant increase in PhN activity contralateral to hemisection of 221.0 ± 4 0.4% (m ± SE); since animals were artificially-ventilated, these changes likely represent an increase in central respiratory drive. These results underscore the state-dependence of crossed bulbophrenic projections and suggest that unanesthetized models may be more sensitive in detecting acute recovery of respiratory output following spinal cord injury (SCI). Additionally, our results may suggest an important role for a group of C1-C2 neurons exhibiting respiratory-related activity, spared by the higher level of hemisection. These units may function as relays of polysynaptic bulbophrenic pathways and/or provide excitatory drive to phrenic motoneurons. Our findings provide a new model for investigating acute respiratory recovery following cervical SCI, the high C1-hemisected unanesthetized decerebrate rat and suggest a centrally-mediated increase in central respiratory drive in response to high cervical SCI.

Enhancing neural activity to drive respiratory plasticity following cervical spinal cord injury

Cervical spinal cord injury (SCI) results in permanent life-altering sensorimotor deficits, among which impaired breathing is one of the most devastating and life-threatening. While clinical and experimental research has revealed that some spontaneous respiratory improvement (functional plasticity) can occur post-SCI, the extent of the recovery is limited and significant deficits persist. Thus, increasing effort is being made to develop therapies that harness and enhance this neuroplastic potential to optimize long-term recovery of breathing in injured individuals. One strategy with demonstrated therapeutic potential is the use of treatments that increase neural and muscular activity (e.g. locomotor training, neural and muscular stimulation) and promote plasticity. With a focus on respiratory function post-SCI, this review will discuss advances in the use of neural interfacing strategies and activity-based treatments, and highlights some recent results from our own research.

Effects of vagotomy on hypoglossal and phrenic responses to hypercapnia in the decerebrate rat.

Hypercapnia characterizes a variety of physiological and pathological states and must be compensated effectively by the respiratory, cardiovascular, renal, and intra- and extracellular pH buffering systems to maintain homeostasis. Several studies have examined the respiratory response to hypercapnia, but contemporaneous changes in respiratory frequency and tidal volume prevent investigating the pure influence on respiratory amplitude. Therefore, we sought to test the effect of hypercapnia on hypoglossal (XII) and phrenic nerve (PN) inspiratory (Insp) and XII pre-inspiratory (pre-I) activities in vagus-intact and vagus-denervated animals. Experiments were performed on six artificially-ventilated unanesthetized pre-collicular decerebrate Sprague-Dawley adult male rats. Vagotomy under normocapnic conditions effected the consistent appearance of significant XII pre-I and a greater increase in XII than PN Insp amplitude. In the vagus-intact state, administration of a hypercapnic (5% CO2, 95% O2) gas mixture resulted in a greater increase in XII than PN Insp activity. In the vagotomized state, hypercapnia caused a drastic increase in XII pre-I and significant non-differential increases in both XII and PN Insp activity. The increase in XII pre-I was significantly greater than hypercapnia-induced increases in XII and PN Insp discharges. Following vagotomy, duration and amplitude of XII pre-I are potently modulated by CO2 tension. Based on our results, we conclude that vagal afferents exert differential inhibition of PN Insp and XII pre-I/Insp motor outputs. The role of vagal control in orchestration and optimization of respiratory response to hypercapnia is discussed.

Inhibition of A5 Neurons Facilitates the Occurrence of REM Sleep-Like Episodes in Urethane-Anesthetized Rats: A New Role for Noradrenergic A5 Neurons?

When rapid eye movement (REM) sleep occurs, noradrenergic cells become silent, with the abolition of activity in locus coeruleus (LC) neurons seen as a key event permissive for the occurrence of REM sleep. However, it is not known whether silencing of other than LC noradrenergic neurons contributes to the generation of REM sleep. In urethane-anesthetized rats, stereotyped REM sleep-like episodes can be repeatedly elicited by injections of the cholinergic agonist, carbachol, into a discrete region of the dorsomedial pons. We used this preparation to test whether inhibition of ventrolateral pontine noradrenergic A5 neurons only, or together with LC neurons, also can elicit REM sleep-like effects. To silence noradrenergic cells, we sequentially injected the α2-adrenergic agonist clonidine (20–40 nl, 0.75 mM) into both A5 regions and then the LC. In two rats, successful bilateral clonidine injections into the A5 region elicited the characteristic REM sleep-like episodes (hippocampal theta rhythm, suppression of hypoglossal nerve activity, reduced respiratory rate). In five rats, bilateral clonidine injections into the A5 region and then into one LC triggered REM sleep-like episodes, and in two rats injections into both A5 and then both LC were needed to elicit the effect. In contrast, in three rats, uni- or bilateral clonidine injections only into the LC had no effect, and clonidine injections placed in another six rats outside of the A5 and/or LC regions were without effect. The REM sleep-like episodes elicited by clonidine had similar magnitude of suppression of hypoglossal nerve activity (by 75%), similar pattern of hippocampal changes, and similar durations (2.5–5.3 min) to the episodes triggered in the same preparation by carbachol injections into the dorsomedial pontine reticular formation. Thus, silencing of A5 cells may importantly enable the occurrence of REM sleep-like episodes, at least under anesthesia. This is a new role for noradrenergic A5 neurons.