Open access peer-reviewed chapter

Hypoxic Preconditioning: The Multiplicity of Central Neurotransmitter Mechanisms and Method of Predicting Its Efficiency

Written By

Elena I. Zakharova, Zanaida I. Storozheva, Andrew T. Proshin, Mikhail Yu. Monakov and Alexander M. Dudchenko

Submitted: 28 March 2018 Reviewed: 15 July 2018 Published: 05 November 2018

DOI: 10.5772/intechopen.80333

From the Edited Volume

Hypoxia and Anoxia

Edited by Kusal K. Das and Mallanagouda Shivanagouda Biradar

Chapter metrics overview

1,240 Chapter Downloads

View Full Metrics


In rats, a single moderate hypobaric hypoxia (HBH) increased the resistance to severe hypoxia (SHBH). The HBH efficiency and neurotransmitter mechanisms of its preconditioning action were investigated by biochemical and pharmacological methods. It will be substantiated in the chapter: (1) HBH preconditioning has its own mechanisms that do not depend on an innate resistance to SHBH and prior hypoxic experience of rats; (2) the same preconditioning effect can be achieved by diverse neuronal pathways and synaptic plasticity means; (3) cholinergic and, presumably, serotoninergic, GABAergic and/or glutamatergic systems of the caudal brainstem, cortex and some other brain structures are involved in HBH realisation; (4) the rate of sensorimotor gating estimated in the model of acoustic startle pre-pulse inhibition (PPI) predicts the efficiency of hypoxic preconditioning and (5) the cholinergic system, including α7 nicotinic receptors, is involved in the mechanisms of HBH-PPI-dependent preconditioning effects.


  • hypoxic preconditioning
  • resistance to severe hypoxia
  • apnoea
  • adaptation to hypoxia
  • mechanisms of hypoxic preconditioning
  • brainstem
  • cortex
  • central neurotransmitter systems
  • pre-pulse inhibition in acoustic sensorimotor startle reaction
  • cholinergic system
  • nicotinic receptors

1. Introduction

1.1. Protective action of moderate hypoxia

It is well known that pathological factors (many poisons, pathogenic viruses) in low doses can initiate protective mechanisms and increase resistance to the corresponding pathologies. Similarly, it is possible to increase resistance to severe hypoxia or ischaemia by exposing the organism or organ to adaptation in conditions of moderate hypoxia [1].

Short-term hypoxic adaptation is a single 1- or 3-h moderate hypoxic continuous or intermittent exposure in which hypoxia alternates with normoxia or hyperoxia [2, 3, 4, 5]. Such hypoxic adaptation is characterized by the mobilization of available cellular reserves and its effect may be manifested within 24 hours [2, 5, 6, 7]. In experimental practice, Murry et al. were the first to describe the protective effect of the short ischaemic exposure against ischaemic stroke, suggested a preconditioning term for this phenomenon [8]. Later, the same authors identified the reperfusion (re-oxygenation) as the second most important adaptive component [9].

Hypoxic factor is the main in ischaemic preconditioning. Therapeutic potential of the hypoxic and ischaemic preconditioning are closely related. Protection from hypoxic damage is also very relevant because the hypoxic component is involved in pathogenesis of many diseases. In vivo, protective action of the hypoxic or ischaemic preconditioning was identified in the diseases of brain [3, 10, 11, 12, 13, 14, 15], heart [8, 16, 17, 18], liver [19, 20, 21] and kidneys [22, 23]. Direct hypoxic preconditioning or drugs that mimic the hypoxic protective response could reveal promising therapeutic targets. Today, understanding of the hypoxic or ischaemic preconditioning mechanisms is a high priority [15].

The brain is central in the problem of hypoxic adaptation not only as the most sensitive organ to hypoxia but also as the coordinator of the functions of all body organs and systems. In the nervous tissue, the functional specificity and individual sensitivity to hypoxia of separate neuronal populations and the corresponding brain structures are of fundamental importance. However, in search of key targets of hypoxic preconditioning, the neuronal mechanisms remain the least studied. It is the aspect of hypoxic preconditioning that is in the centre of our attention. On the other hand, the neuronal autonomous mechanisms of respiration and blood circulation in the norm and under severe hypoxic conditions are intensively studied. These data serve as a serious help in the analysis of neuronal mechanisms of hypoxic preconditioning.

Another important problem that we have tried to solve is the methodology for studying the mechanisms of hypoxic preconditioning.


2. Systemic reaction of autonomic systems to hypoxia

The primary and immediate response to hypoxia is always recorded in the autonomic respiratory and cardiovascular systems. Central representation, the neurons of both systems, is located in the medulla oblongata and pons Varolii (caudal brainstem) and spinal cord. Autonomic systems are functionally closely interrelated by the “respiratory centre”, groups of respiratory neurons, which support respiratory rhythm [24, 25, 26].

It has been shown that the “hypoxic” response involves activation of the autonomic sympathoexcitatory reticulospinal pathways, primarily from the peripheral or central chemoreceptors and the stimulation of respiration, heart activity and blood circulation aimed at restoring the blood level of O2 and CO2 exchange and pH [27, 28, 29, 30, 31, 32].

The sequence and development of hypoxia-induced events are carefully researched [30, 31]. In general, this systemic response is a result of the wide cooperation of different functional groups of neurons of the central autonomic respiratory and cardiovascular systems: sensory, primary and secondary chemo and baroreceptors of the nucleus tractus solitary (NTS) and ventrolateral medulla (VLM); relay neurons; reticulospinal neurons (area C1 of the rostral VLM); efferent neurons of the preganglious parasympathetic nuclei of cranial nerves and spinal motoneurons.

Another important adaptive express response to hypoxia is non-sympathetic activation of the cerebral blood flow that is based on the redistribution of blood flow towards the brain [31, 33, 34, 35, 36]. Two centres have been detected: the “parasympathetic cerebrovasodilator centre” or in another way “dorsal facial area” (DFA) and the “medullary cerebral vasodilator area” (MCVA). Both centres are located in the rostrolateral part of medulla oblongata (and DFA also partially covers the pons Varolii), and their innervation, including hypoxia, initiates elevation of the cerebral blood flow by parasympathetic [33, 37, 38] or relay cerebripetal pathways [34, 39]. Also, the connection of MCVA with the sympathetic vasomotor mechanisms is shown: in the sympathoexcitatory zone C1, the presence of the O2-sensitive neurons of which activation excites the cerebripetal pathway; dependence of the cerebrovascular MCVA efficiency of safety of the reticulospinal pathway and existence of collaterals from the sympathoexcitatory neurons in the cerebripetal direction [34, 39].

All of these systemic reactions are revealed in cats [40] and respiratory reactions from those in rats [36, 41, 42] with high, intermediate and low resistance to very severe hypoxia (3% O2). Differences between these groups were mainly in expression and duration of the responses before apnoea. Under moderate hypoxia, all the compensatory reactions, including cerebral blood flow elevation, are maintained during the whole session of hypoxic training in rats [36, 43, 44], and all of them are the physiological basis of hypoxic preconditioning [44, 45, 46].

It should also be noted that the structures of the forebrain are the most unstable to ischaemic/hypoxic injuries [47], and the most interesting have been shown to be the cortex and hippocampus, as these are the higher brain structures responsible for cognitive functions. Both the cortex and hippocampus interact with the cardiorespiratory systems, participating in the regulation of voluntary respiration and hypothetically adaptive reactions of the respiratory and cardiovascular systems [25, 26, 48, 49].

But we assumed that under moderate hypoxia conditions (10–12% O2 for rat), the key role in the preconditioning belongs to the autonomic systems.


3. Autonomous regulation of respiration: overview of neuronal populations responsible for generation of apnoea

In our experiments on rats, the preconditioning effect of moderate hypoxia was evaluated under conditions of severe hypoxia by the time (T) until agonal inspiration (apnoea).

Thus, among the key neurotransmitters of caudal brainstem are of interest, involved in the generation of apnoea. The most studied in this respect are inhibitory neuromediators or neuromodulators of the opioid, serotoninergic, GABAergic, glycinergic and adenosinergic systems. And we will certainly touch upon the cholinergic system as an object of our research, which, according to our data, occupies not the last place in the preconditioning mechanisms.

In the overview, special attention was paid to the synaptic transmission; since in our studies, we evaluated the response of the synaptic pool of the caudal brainstem and some other brain structures.

3.1. Opioid system

Opioids cause apnoea selectively through μ-receptors. The action of the majority of opioid analgesics is associated with the stimulation of μ-receptor type. However, μ-receptor agonists cause side effects, among them respiratory depression. It was shown in cats and rats that μ-receptor agonists morphine and/or fentamine depressed respiration, initiated central apnoea or apneusis breathing [50, 51, 52, 53, 54]. μ-Receptors are widely distributed in the brain, but their mechanisms of action and targets are still poorly understood. According to some data, Bötzinger Complex (BötC) and especially pre-Bötzinger Complex (preBötC) are responsible for opioid-initiated destruction of respiration [51, 52]. According to other data, such opioid-sensitive sites are numerous in the brainstem [53]. An endogenous ligand of μ-receptors is β-endorphin. However, endorphinergic fibres or terminals in the caudal brainstem have not been described to date. Hormonal mode of distribution of endorphins through the blood is well known. In consideration of the chemical stability of the ligands of the opiate receptors, it is assumed that they penetrate into the respiratory centre through the cerebrospinal fluid [25].

3.2. Serotoninergic system

I.v. administration of serotonin (5-hydroxytryptamine, 5-HT) or 5-HT3 receptor agonist phenylbiguanide provoked “von Bezold-Jarisch” or C-fibres reflex (bradycardia, drop in blood pressure, apnoea) passing, the authors believe, through 5-HT3 receptors in the nucleus tractus solitary NTS [55]. Really, bradycardia from the triad of Bezold-Jarisch reflex was potentiated by the i.c. administration of phenylbiguanide and was dose-dependently weakened by the i.c. administration of receptor antagonist granisetron [55]. Granisetron microinjected into NTS significantly attenuated both bradycardia and hypotension [55].

Under normoxic conditions (cat), both i.v. administration and microinjection into preBötC of 5-HT1A receptor agonist 8-OH-DPAT produced apnoea and arrested respiratory neuronal activity [32]. Previously, it has been found that stimulation of the nucleus raphe obscurus provoked that apnoea and 5-HT1A receptors, which are abundantly expressed in the respiratory neurons of ventral respiratory group, were involved in this mechanism [56].

Under hypoxic conditions (cat), using microdialysis and registration of the phrenic nerve and respiratory neurons of the ventral respiratory group activity, it was shown that elevation of 5-HT levels in the extracellular space of the ventral respiratory group clearly coincided with the beginning of hypoxic depression and apnoea (5–10% O2) [32]. The authors revealed that such high correlation with hypoxic depression was selective for 5-HT in this respiratory region because it was absent with the levels of other investigated mediators or modulators (GABA, glutamate and adenosine). In the same study, microinjection of 8-OH-DPAT into preBötC on the apneustic patterns background, initiated by prolonged moderate hypoxia (cat, 15% O2), resulted in normal respiratory parameters. In contrast to the 8-OH-DPAT effects, blockade of 5-HT1A receptors during hypoxia by antagonist NAN-190 resulted in dramatic enhancement of apneustic inspiratory activity patterns.

The molecular signalling pathway was later traced through these receptors on the glycinergic respiratory neurons of preBötC and, possibly, neighbouring regions of the ventral respiratory group [51]. The activation of 5-HTR1A potentiated glycinergic currents in all postsynaptic neurons receiving glycinergic inputs through glycine alpha3 receptors (GlyRalpha3) that not only excitatory (glutamatergic) but also inhibitory (glycinergic) neurons and enhanced their inhibition. It is proved that the 5-HTR1A-GlyRalpha3 signalling pathway can restore the respiratory circuitry and disturbed by hypoxia or some other factors (opioid intoxication) [26, 51].

In the rat, the long deep apnoea occurs when stimulation of special neurons within cluster of the serotoninergic neurons in the medullary raphe nuclei (the raphe pallidus, magnus and obscurus) [57]. The point of these neurons is called the “midline apneic site” (MAS) and suggested their 5-HTergic nature. By the morphoimmunological methods, the same researchers proved the relationship of MAS with many higher brain regions and some areas of the medulla oblongata, including the ventral respiratory group [58].

It was also revealed the action of 5-HT on the cerebral circulation. Intravenously (i.v.) injections of 5-HT (cat, rat) may have different regional actions on the brain blood vessels of various categories, but its decisive value was the development of cerebrovascular constriction, a decrease in the rate of cerebral blood flow and a drop in blood pressure [59], which was enhanced by ischaemic exposure [60]. All cerebrovascular reactions appeared similar or were more pronounced at i.c.v. injections (cat) or the application of 5-HT on the brain (rat) indicating the central nature of its actions [59]. It has been proven the central action of 5-HT in the DFA on the cerebral circulation. Injected into DFA, 5-HT or alaproclate, a 5-HT reuptake inhibitor, synaptically inhibited the glutamatergic activation of the parasympathetic preganglionic cholinergic motoneurons and thus reduced the rate of blood flow in the common carotid arteries [38]. Also, by i.v. administration of 5-HT, a significant drop in the rate of the cerebral circulation was revealed in the cortical parietal area as well as in the frequency and depth of breathing [61]. The drop in the rate of the cerebral circulation coincided with the accumulation of CO2 in the blood. It should be noted that some serotoninergic neurons in the dorsal raphe nucleus (in the midbrain), connected with MAS [58], have CO2/pH chemoreception and deep hypercapnia (9% CO2) produced an increase in their firing rate [62].

These data suggest that the reduction of the serotoninergic influences on, presumably, any site of the autonomous cardiorespiratory regulation, except the ventral respiratory group facilitates breathing and/or conduces to a delay of generation of apnoea.

3.3. GABAergic system

GABA side by side with glutamate is the most widespread mediator in the central nervous system and is involved virtually in all nervous processes. Central GABAergic effects on the cardiorespiratory functions are not unidirectional. GABA is a principal inhibitory neurotransmitter of the sympathoexcitatory and baroreflex sympathoinhibitory glutamatergic pathways from peripheral chemo- and baroreceptors through the second-order sensitive neurons of NTS [63, 64]. The GABAergic neurons of caudal VLM are interneurons in the baroreceptor reflex arc and directly inhibit the sympathoexcitatory C1 zone neurons of rostral VLM [64].

In the NTS (rat), the agonist of GABAB receptor baclofen attenuated the cardiorespiratory reflexes of C-fibres, provoked by phenylbiguanide, bradycardia and decrease in frequency of breathing with no effect on hypotension and apnoea when microinjected into any point of dorsomedial NTS in dose of 60 pmol [65]. When it was injected into the inhibitory zone of the dorsal respiratory group of NTS only (0.5–0.6 mm caudal to the obex [66]), baclofen removed C-fibre-provoked apnoea and the antagonist of GABAB receptor CGP 35348 (2.8 nmol) newly restored it. Similar effect was obtained by the systemic administration of high doses of the GABAB receptor agonists hydroxybutyrate and phenibut (6.9 mmol/kg, i.v., and 2.3 mmol/kg, I.P., respectively, rat) [67, 68]. Both agonists attenuated the decrease in the frequency of breathing and abolished or shortened the duration of apnoea of C-fibres reflex, which are provoked by 5-HT. Hydroxybutyrate (i.v.) and phenibut (I.P.) caused a complete loss of sensitivity of the respiratory system to vagotomy are supposed by central blocking transmission of afferent impulses from the baroreceptors of lungs and airways to the second-order barosensitive neurons in NTS [54, 67].

Under the normoxic conditions, baclofen also prolonged the inspiration and increased the heart rates injected into NTS in the same dose of 60 pmol in rats [65]. In the high doses in cats, in hundreds nmoles for baclofen and micromoles for GABA and sodium hydroxybutyrate, the decrease in respiratory frequency and apneusis breathing arose in the majority of intact animals when GABA or the GABAB receptor agonist was microinjected into the dorsal respiratory group region (ventrolateral NTS) [69]. The same respiratory reactions were obtained after the i.v. administration of hydroxybutyrate in cats and rats [25, 69] and after the I.P. administration of phenibut in rats [54].

Under hypoxic conditions, similar to our HBH (10% O2, 45–50 minutes, rat), participation of GABA in respiratory mechanisms of hypoxia was investigated [43]. Moderate hypoxia initiated a primary pronounced ventilatory increase (minute respiratory volume) followed by a gradually decline to a second-stable level above pre-hypoxic level and a sustained increase in respiratory frequency during the hypoxic exposure. Under these conditions, GABA had a depressant effect on the ventilation. By in vivo microdialysis, the elevation of GABA concentration in NTS coincided with the ventilatory decline. By microinjections into sensitive non-apnoeic region of NTS of agonists and antagonists of GABAA and GABAB receptors, both the agonists muscimol (150 pmol) and baclofen (400 pmol) were injected 10 minutes before the hypoxic exposure significantly attenuated the early increase of ventilation, and on the contrary, the antagonists of GABAA receptor bicuculline and of GABAB receptors saclofen (400 pmol) and CGP-35348 (2.5 nmol) in the 40-minute hypoxic exposure abolished the late ventilatory decline and reduced the GABA elevation [43]. The authors have shown that for GABA activation in NTS, peripheral chemoreceptor stimulation is essential because it is the normoxia or under denervation of the carotid body, GABA level in the NTS did not change and the effects of GABA antagonists did not appear.

Another study also showed that endogenous GABA in the NTS inhibits the carotid chemoreflex (rat) [70]. Microinjection of the selective GABA uptake inhibitor nipecotic acid into the commissural sub-nucleus of NTS attenuated the increases in respiration and elevation in arterial blood pressure elicited by carotid chemoreceptor stimulation. These effects were completely antagonised by the GABAA antagonist bicuculline (20 pmol) but not by the GABAB antagonist saclofen (400 pmol), injected into the same site.

I.v. administration of GABAA receptor antagonist picrotoxin enhanced ventilation through an increase in respiratory frequency and minute tidal volume (rat) [42]. A severe hypoxia (3% O2, rat) in the first few minutes, like in the moderate hypoxia, initiated the same dynamics in the increase of minute tidal volume and respiratory frequency [40, 41, 42], and picrotoxin (i.v.) significantly potentiated the activation of both respiratory functions (cat and rat) [42]. Also, the authors observed that these effects of picrotoxin were most pronounced in the high resistance rats compared with low and intermediate resistance rats. Moreover, it was found that under these hypoxic conditions, picrotoxin greatly extended the time before apnoea in all resistance rat groups [42].

Note that these data indicate that systemic administration of the GABA receptor agonists and antagonists reflects the central action of these drugs within NTS.

In the ventral respiratory group of VLM, GABAergic neurons have other effects and can have a protective action on respiration under hypoxic conditions. By in vivo microdialysis, the level of GABA (and glutamate) in the respiratory region of VLM increased transiently during early periods of severe hypoxia (5–10% O2, cat), coinciding with augmented phrenic nerve activity and fell below the control levels during central apnoea [32]. The authors suggest that GABA may be important for regulation of level of enhanced respiratory network activity at the onset of hypoxia. In addition, in BötC and preBötC of VLM, the pacemaker nature of some GABAergic respiratory types of neurons is assumed [26, 71]. Microinjections of GABA into BötC facilitated respiration (increased the tidal volume) and into preBötC significantly inhibited respiration (reduced the tidal volume) [72]. The same lack of uniformity was observed under the action on GABA receptors of B and A subtypes at BötC/preBötC.

Participation of GABAB receptors in preBötC and BötC respirator functions was revealed using agonist baclofen. Under the normoxic conditions, the influences through GABAB were directed towards the respiratory depression when baclofen was administered into both BötC and preBötC [73] or BötC only and, on the contrary, towards the weak respiratory stimulation when it was microinjected into preBötC [72]. These two studies were performed on rabbits (first) and rats (second). However, we believe that the main difference was in doses. It seems that baclofen is more selective at 15–25 times smaller dose (2 pmol) [72] and therefore caused the opposite effects and influenced the breathing of GABAB receptors in BötC, which was more expressed than in preBötC. At the same time, it should be noted that GABAB receptor stimulation by baclofen at BötC suppressed breathing in both doses.

The blockade of GABAA receptors within these respiratory complexes by the antagonist bicuculline or gabazine disturbed respiration until apnoea [71, 73, 74]. Blocking GABAA receptors by picrotoxin also disturbed respiratory rhythm and provoked apneusis breathing when it was injected into the fourth ventricle [42]. At the same time, microinjections of bicuculline into preBötC recovered the respiration against the background of apnoea caused by the blockade of GABAA receptors in BötC [73].

Thus, the natural reduction of GABAergic synapses would help the delay of generation of apnoea by cutting back the impacts of GABA in the sympathoexcitatory C1 zone neurons of rostral VLM, through both GABAA and GABAB receptors in the sympathoexcitatory conductive sensory pathways and dorsal respiratory group within NTS and through at least GABAB receptors in BötC and possibly GABAA receptors in preBötC within VLM.

3.4. Glycinergic system

Glycinergic neurons of the medulla oblongata were identified in the ventral respiratory group. In BötC and especially preBötC, glycinergic neurons are more than half of all respiratory neurons [74]. It was shown in vivo that many of them generate the respiratory activity, that is, pacemakers [71]. Both in preBötC and in BötC, the blockade of glycine receptor by the antagonist strychnine resulted in the suppression of respiratory activity until apnoea [26, 51, 73] or disturbance of the respiratory cycle, the modification of activity of the post-inspiratory neurons and, as a consequence, the transfer of the normal three-phase cycle into the pathological biphasic [26, 75]. These data indicate that the inhibitory effects via glycine receptors are required for normal respiratory function.

By other data [76], glycine is also required for normal respiratory function in the dorsal respiratory group. In the intermediate sub-nucleus of NTS, the secondary barosensitive neurons receive glutamatergic afferent inputs from the pulmonary rapidly adapting stretch receptors and have inhibitory influences on respiration. The authors found that these secondary neurons receive the phasically acting inputs from glycinergic neurons, which inhibit their activity in the inspiratory phase.

In another networks of the medulla oblongata, participation of glycine was identified as a sympathoexcitatory or sympathoinhibitory modulator. Sympathoinhibitory influence on glutamate pathway from caudal to rostral VLM was mediated by glycine in a manner independent of GABAA and GABAB receptors [77]. Microinjections of glycine into NTS decreased arterial pressure and heart rate [78], inhibited the pressor but not bradycardic responses produced by L-glutamate microinjection in the same site [79] and, at the same time, inhibited the depressor and bradycardic responses to L-glutamate [78].

Thus, on the certain sites of the glutamatergic pathways of VLM and NTS, attenuation of the glycinergic influences may contribute to the delay of apnoea generation.

3.5. Adenosinergic system

Adenosine is present in the CNS at pharmacologically active concentrations [80, 81, 82], and it is now recognised as a neuromodulator. The extracellular concentration of adenosine in the brain increases dramatically during hypoxia or ischaemia [32, 80, 83]. Adenosine has a depressor effect on the neuronal activity through A1 and A3 receptor types and antagonistic effect through A2 receptors [83, 84]. Autoradiographic and immunohistochemical studies illustrate the presence of A1 and A2 (mainly A2a) binding sites/receptors in NTS, VLM and other brainstem regions that are important in cardiorespiratory control [81, 85].

The presence of the enzyme 5′-nucleotidase, which converts AMP to adenosine in the fractions of synaptosomes (cortex and hippocampus, rat) [86] and adenosine in the fraction of the synaptic vesicles (rat brain) [87], indicates the existence of adenosinergic pre-synapses in the brain. In addition, a high-affinity transport system for adenosine and adenosine deaminase, an enzyme of adenosine cleavage in the synaptic cleft, was revealed in the crude synaptosomal fraction of NTS (rat brain) [88, 89].

In the caudal brainstem, adenosine and as a rule, the following selective agonists and antagonists of A1 and A2a adenosine receptors used to study the role of adenosine in the regulation of cardiorespiratory functions: the agonist N6-cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) of A1 receptors; the agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5’-N-ethylcarboxamidoade (CGS 21680) and antagonists 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo(1,5-c)quinazoline-5-imine (CGS 15943A) and 4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM 241385) of A2a receptors. Also, the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT) and A1/A2 receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT) with higher affinity for A1 receptors [85] were applied.

In the chemo- and baroreceptive sites of the NTS, opposite effects on cardiovascular parameters were revealed under the action of these two types of receptors in the intact rat brain. Microinjection into NTS of A1 receptor agonist CPA stimulated and of A2a receptor agonist CGS21680 decreased the blood pressure, and both agonists provoked bradycardia [82, 85, 90, 91, 92]. It was found that the opposite effects of A1 and A2a receptor agonists on blood pressure are due to the action on the glutamatergic afferent nerve fibres from arterial and pulmonary baroreceptors. Accordingly, the stimulation of A1 receptors inhibited glutamate release, and the stimulation of A2a receptors activated it [91, 92]. The effects of А2а receptors considerably dominated over A1 receptors in NTS [82, 90, 91], and the decrease of blood pressure in all of these studies was removed by the antagonists of A2a receptor CGS15943A or ZM241385 and of A1 receptors agonist DPCPX, while bradycardia was selectively antagonised by CGS15943A but not by DPCPX. The same agonist of A1 receptors CPA had the hypotensive action followed by microinjections into NTS at doses far exceeding those used in the cited studies and was antagonised by i.v. infusions of A1 receptor antagonist DPCPX [93].

From the above data, under certain conditions of the disturbance of cardiorespiratory functions, the NTS A2a receptors in concert with A1 receptors are involved in the activation of the sympathetic functions. At the same time, the decrease of synaptic influences on the A2a receptors and possibly on the A1 receptors of NTS could contribute to the delay of apnoea generation.

Stimulation of 5HT3 receptors by agonist phenylbiguanide (i.v. injection) initiated “cardiopulmonary chemoreflex” (hypotension, bradycardia and apnoea) [82, 94], aka reflex of C-fibres or von Bezold-Jarisch in other sources. This reflex was attenuated/blockaded or conversely potentiated by microinjections into NTS of the A2a receptor agonist CGS21680 or antagonist ZM241385, respectively [82]. The same reflex, when developed under a severe haemorrhage model, was inhibited by endogenous adenosine; this inhibition was removed by microinjections into NTS of the antagonist 8-SPT [82]. The authors suggested that A2a receptors were responsible for the activation of inhibitory influence on the “cardiopulmonary chemoreflex” pathway and later proved a participation of GABAA and much weaker GABAB receptors in this mechanism within NTS [94].

The role of А1 receptors in the cardiorespiratory functions was revealed in the VLM, where this receptor type had the highest density [85]. In the rostral VLM (rat), microinjections of adenosine into the pressor zone C1 augmented the sympathoexcitatory reflex of increase in blood pressure evoked by electrical stimulation of the “hypothalamic defence area”and, on the contrary, the microinjections of 8-SPT into C1 or both peripheral and central injections of the selective A1 receptor antagonist DPCPX this reflex reduced [80]. Vice versa, microinjections of adenosine into the ventral respiratory group (cat), acting on pre- and postsynaptic A1 receptors, led to the depression of spontaneous and stimulus-evoked synaptic activity of the respiratory neurons and to the fall of mean respiratory drive potentials. The depressive effects of adenosine were abolished after the i.v. administration of the antagonist DPCPX [95].

Under severe hypoxia (5–10% O2, cat), i.v. DPCPX administration retained the same direction of action on respiration. This A1 antagonist showed marked protective properties, namely preventing the early hypoxic depression of stimulus-evoked activity of the respiratory neurons in the ventral respiratory group, significantly delayed the onset of apnoea and reduced the recovery time [95]. In the same hypoxic conditions in the ventral respiratory group using microdialysis, an intensified increase of a level of the endogenous extracellular adenosine was identified, and this reaction was developed in the background of the apnoea after it began [32]. The authors noted that the hypoxic release of adenosine was occurred surprisingly late than hypoxic depression of respiration and cannot be responsible for the onset of the hypoxic depression of respiratory neurons, and that it is noteworthy that increases in adenosine levels outlasted hypoxic periods, which were not associated with pronounced depression of phrenic nerve activity.

However, endogenous adenosine may be involved in the mechanism of a secondary suppression of the hypoxic activation of the cerebral blood flow. As mentioned above, the fall in the rate of cerebral blood flow under the von Bezold-Jarisch reflex conditions also occurred after the apnoea beginning in the background of its end and coincided with the accumulation of CO2 in the blood [61]. Under severe hypoxia (8% O2, rat), i.v. administration of the non-selective antagonist 8-PT, penetrating through the blood-brain barrier unlike not penetrating 8-SPT, potentiated hypoxic alkalosis and hypocapnoea that arise from the initial hyperventilatory response, extended the increase in tidal volume and heart rate, reduced the decrease in arterial pressure, stopped the progressive increase of the carotid vascular conductance and, at the same time, showed a pronounced tendency for cerebral blood flow to be better maintained during hypoxia [31, 96]. The authors proved that all effects of 8-PT were central and were a consequence of the influence of the antagonist on the secondary fall in ventilation.

Taken together, under hypoxia, endogenous adenosine of the ventral respiratory group can contribute to the fall in the ventilation and, as a consequence, initiate hypercapnia. Accordingly, attenuation of these effects of adenosine can contribute to maintaining the cerebral blood flow.

3.6. Glutamatergic system

It should also be added that apnoea can be also caused by microinjection of the main excitatory neurotransmitter glutamate into certain sites of the medulla. Therefore, the neurons of the raphe nuclei were stimulated by glutamate for apnoea in MAS [57]. Also, apnoea occurred when glutamate was microinjected into the inhibitory site of the respiratory neurons of the dorsal group of NTS (behind the obex) [66]. Apparently, it is the action of the described above glutamate afferents triggering baroreflex [64, 78]. In NTS, these afferents switched on bulbar interneurons, mainly glutamatergic, which, as shown, transfer drive signals to the parasympathetic motor nucleus ambiguous and stimulate bronchoconstrictor reflex [97] and also through the caudal VLM, having an inhibitory action on the sympathoexcitatory reflex [64].

3.7. Cholinergic system

Starting from Loeschcke studies [27, 98], the central effects of ACh and its analogues on the respiration and blood circulation are intensively investigated. As for many other neurotransmitters, the cholinergic participation is detected in the majority of functional sites of cardiorespiratory networks as well as the ambiguity of the cholinergic effects depending on drug dose, application site and reception [39, 99].

Caudal brainstem structures include several cholinergic sources: (1) projections from the reticular formation of the midbrain tegmentum [100, 101, 102, 103]; (2) afferents of the nodose ganglion sensory neurons from the lung mechanoreceptors to the NTS [97, 104, 105] and (3) the neurons of pons Varolii and medulla oblongata, including reticular areas, NTS and efferent parasympathetic preganglionic neurons of the motor cranial nerves nuclei [99, 102, 103, 105, 106, 107].

Concerning the connections and functional effects of the cholinergic system of the caudal brainstem in response to hypoxia, we recently published an overview similar to the above [108, 109].


4. Preconditioning effects on resistance to severe hypoxia and synaptic pool of caudal brainstem, cortex and hippocampus

The sub-chapter briefly describes our experimental approaches on the study of neuronal mechanisms of hypoxic preconditioning. During the planning of experiments, we were guided by the data that the short-term adaptation, especially after continuous hypobaric hypoxia, had the pronounced and rapid preconditioning effect in the first minute of re-oxygenation [2, 6, 7].

The experiments were carried out according to two protocols.

4.1. General experimental conditions and procedures

Animals. The male outbred albino rats aged 2–2.5 months (200–250 g) at the beginning of the studies. All animal care and experimental procedures were conducted in accordance with the official regulations of the European Communities Council Directive on the use of laboratory animals of November 24, 1986 (86/609/EEC).

Hypoxic models. Hypoxic preconditioning, the continuous hypobaric hypoxia (HBH): an altitude of 5000 m (11% O2), 60 minutes. Test for resistance to hypoxia, severe hypobaric hypoxia (SHBH): the critical altitude of 11,500 m (4.5% O2). In the latter case, resistance to hypoxia was recorded with respect to time (T) until agonal inspiration (apnoea) in combination with a loss of voluntary control of body tone. Apnoea was a defining attribute.

Re-oxygenation after HBH. Four minutes.

Brain structures for biochemical investigations. The caudal brainstem, cortex and hippocampus.

Preparative methods for biochemical investigations. From each brain structure, the sub-fractions of synaptic membrane and synaptoplasma were isolated from the fractions of “light” and “heavy” synaptosomes by routine preparative methods using discontinuous sucrose gradients.

The sub-synaptic level of fractionation made it possible to study the largest functionally different pre-synaptic compartments, and it was very informative. Moreover, synaptic membrane sub-fractions were considerably cleaned from glial, mitochondrial and free (not docked) synaptic vesicle contaminations.

Analytical methods. In the sub-synaptic fractions, the choline acetyltransferase (ChAT, functional marker of cholinergic neurons) activity by radiometric method [110] and protein content by spectrophotometric method [111] were assayed.

For details of the experimental procedures, see [112, 113, 114].

4.2. Experimental protocol number 1

It is important to bear in mind that animals (and humans) are very different in their resistance to severe hypoxia, and this implied different mechanisms. Because of this, since the publication of Purshottam and Ghosh [115], animals were divided into resistance to severe hypoxia, using pre-testing them under the same hypoxic conditions [116, 117, 118]. Later, the pre-testing under severe hypoxia was applied to rats for the investigation of mechanisms of hypoxic preconditioning [2, 7] and in our experiments [108, 109, 113, 119].

So, in our experiments (Figure 1) in each sample, most of the rats were pre-tested under SHBH and divided into groups of low, high and intermediate resistance to hypoxia with T1 < 3.5 minutes, T1 > 7 minutes and between them, respectively. For the following 4–5 weeks, all pre-testing rats were kept under standard vivarium conditions after which the rats in each pre-tested group and the rats in not pre-tested group (intact group) were sub-divided into experimental (HBH) and control groups, and the rats of all experimental groups were subjected to a single HBH session. Four minutes after the end of HBH, the rats from each experimental group, which were subjected to SHBH and T2 (or T1 in intact group), were estimated or taken in the biochemical experiment. The control groups underwent all the procedures after HBH simultaneously with the corresponding experimental groups.

Figure 1.

Scheme of experimental protocol number 1.

4.3. Experimental protocol number 2

In these experiments, the pre-testing under SHBH was excluded. Instead, all rats were pre-tested in the model of acoustic sensorimotor startle reaction, and the magnitude of pre-pulse inhibition (PPI) was estimated [120]. Two to four days after pre-testing, the experimental rats were subjected to a single HBH session, and 4 minutes after the end of HBH were subjected to SHBH (as in Scheme number 1). The control rats underwent all the same procedures except HBH.

Using this experimental scheme, pharmacological experiments were also carried out (Figure 2). The cholinergic nicotinic mechanisms of HBH preconditioning were investigated using the selective agonists of nicotinic receptors (nAChRs) α4β2 type metanicotine RJR 2304 (RJR) and α7 type PNU-282,987 (PNU, Tocris Bioscience, Bristol, UK for both agonists) and a bipolar aprotic solvent for PNU dimethyl sulfoxide (DMSO, LLC “Tula Pharmaceutical Factory”, Tula, RF).

Figure 2.

Scheme of experimental protocol number 2.

Rats in the RJR group received a single I.P. injection of RJR (26 nmol/kg, n = 8) in the physiological saline. Rats in the PNU group received a single I.P. injection of PNU (26 nmol/kg, n = 23, or 260 nmol/kg, n = 12) in 3% DMSO. Rats in the DMSO group received a single I.P. injection of 3% DMSO (n = 16). Rats in the HBH group received a single I.P. injection of the saline (n = 23). Both drugs, DMSO and saline, were injected 10–15 minutes before HBH session.


5. Experiments on the protocol number 1

5.1. The effect of HBH on the resistance of rats to SHBH

HBH markedly increased the mean values of the resistance of rats to SHBH in all the investigated groups (Figure 3). After HBH session, all rat groups showed a similar range of values for resistance to SHBH. In fact, the T values of these groups formed the same variational series (Figure 4) [113].

Figure 3.

Preconditioning effects of HBH on the resistance to SHBH of the low-resistant (A), intermediate-resistant (B), high-resistant (C) and intact (D) rats. T values, a time before apnoea, are expressed as means ± SE. For each group of bars: grey bars, T2 values in the control pre-tested rat groups (A, B, C; n = 12, 9, 14, respectively) and T1 value in the control intact rat group (D; n = 18); light bars, T2 (A, B, C) or T1 (D) values in the corresponding HBH groups (n = 11, 8, 10 and 19 in A, B, C and D, respectively). **p < 0.025 compared to the respective control, Fisher’s exact test.

Figure 4.

Individual T values of the rat resistance to SHBH after HBH in the low-resistant (A), intermediate-resistant (B), high-resistant (C) and intact (D) rat groups. Resistance to SHBH demonstrates that T values of all HBH groups formed the same variational series. n = 11, 8, 10 and 19 in A, B, C and D as in the corresponding HBH groups in Figure 3.

In biochemical experiments, the reaction on HBH of synaptic pool of caudal brainstem and cortex (no reaction was shown in the hippocampus) in the low- and high-resistant rats and intact rats showed that the same preconditioning hypoxic effect can be achieved by various neuronal pathways and plastic synaptic tools. For a detailed analysis, see [108, 109, 112]. Briefly, it was revealed in the following.

5.1.1. In the low-resistant rats

In the low-resistant rats, in the caudal brainstem (Figure 5a), the inhibition of water-soluble ChAT activity in the pre-synapses of heavy synaptosomal fraction corresponded to the functional characterisation of subtypes of the lung barosensitive C-fibres conducting afferentation to NTS through the nodose ganglion [121, 122]. It is known that apnoea is often preceded by the classic reflex of C-fibres (frequent shallow breathing, bradycardia and hypotension) [65, 122, 123]. We substantiated that the cholinergic C-fibres could act on nAChRs affecting theirs through secondary cholinergic barosensitive neurons and the weakening of their influences led to the suppression of parasympathetic reflexes occurring in NTS and thereby to the augmentation of resistance to SHBH.

Figure 5.

The effect of a single HBH session on the ChAT activity (A) and protein content (B) in the sub-synaptic fractions of the caudal brainstem in the pre-tested low-resistant (a) and high-resistant (b) rat groups and in the intact rat group (c). The values of ChAT activity and protein content are expressed as means ± SE. (C) Sub-fractions of light synaptosomes; (D) sub-fractions of heavy synaptosomes. In each pair of bars: left (dark) bar, sub-fraction of synaptic membranes; right (light) bar, sub-fraction of synaptoplasm. The data are shown as percentages as compared to the control, which was taken as 100%. *p < 0.05; **p < 0.025, Fisher’s exact test.


5.1.2. In the high-resistant rats

In the high-resistant rats, in the caudal brainstem (Figure 5b), HBH provoked inhibition of the water-soluble ChAT activity in the pre-synapses of light synaptosomal fraction. We substantiated that the nerve endings of this rat group may be outside NTS. Additionally, a correlation was found between the HBH-induced changes in activity of water-soluble ChAT in caudal brainstem and membrane-bound ChAT in pre-synapses of cortical projection neurons (Figure 6b, the cortical light synaptosomal fraction [124, 125]) (r = +0.911, p < 0.02, n = 6, Pearson’s correlative test). This allowed us to assume that the inhibition of the water-soluble ChAT activity under HBH conditions in this group of rats occurred in pre-synapses of the projection neurons from laterodorsal (LDT) and/or pedunculopontine (PPT) tegmental cholinergic nuclei of the middle brain. LDT and more intensively PPT send plurality of the fibres to both the pontine and the medulla oblongata nuclei [101, 102, 126] and also to the cortical cholinergic projection neurons of the basal forebrain nuclei [126].

Figure 6.

The effect of a single HBH session on the ChAT activity (A) and protein content (B) in the sub-synaptic fractions of the cortex in the pre-tested low-resistant (a) and high-resistant (b) rat groups and in the intact rat group (c). Designations are as shown in Figure 5.

In the high-resistant rats, in the caudal brainstem (Figure 5b), a simultaneous decrease in the content of synaptic c- and m-proteins (r = +0.871, p < 0.05, n = 6) in the heavy fraction in non-cholinergic pre-synapses (correlation between cChAT activity and c-protein content is absent) suggests the possibility of reduction of the number of synapses in non-cholinergic neurons.

According to the literary data in sub-chapter 3, in the first place, the serotoninergic system is reported to be involved in the provocation of apnoea in all of the studied key areas of the autonomic regulation of the cardiorespiratory functions. At the same time, the reduction of the influences of any analysed neurotransmitter systems at corresponding sites may be involved in the mechanisms of the hypoxic preconditioning. It was therefore necessary to analyse the presence of pre-synapses of these neurotransmitter systems in the heavy fraction of synaptosomes, and their representation in the fraction must be enough to identify their reduction by means of such non-specific parameters as protein content. Mediator composition of the heavy fraction of synaptosomes

Analysis of the synaptosomes with the above mediator specificity showed that, as expected, synaptosomes with any mediatory specificity have a wide range of density and sizes. This is shown for glycine, glutamate, serotonin and GABAergic pre-synapses under fractionation in continuous sucrose density gradient [127, 128, 129]. Also, in discontinuous sucrose density gradient, serotonin, glutamate and GABAergic pre-synapses were revealed in the light and heavy fractions of synaptosomes [130, 131, 132, 133, 134, 135].

In sucrose-percoll gradient, the adenosinergic pre-synapses were isolated in the percoll interlayer 10–16% [86]. This percoll fraction apparently corresponds to the heavy fraction of synaptosomes in the sucrose gradient. This is indicated by a similarity in size of synaptosomes ([136, 137] compared with our data [124]) and a significant percentage of the free mitochondria in the fractions. It is known that the density of a substantial part of the free mitochondria coincides with the density of synaptosomes with an expressed vector of the concentration of the mitochondrial organelles towards denser layers of sucrose [128]. As a result, the mitochondria are present in large numbers in the heavy fraction of synaptosomes (20–40% and more) [124, 134], while they were revealed only as the single irregular inclusions in the light fraction [124, 134, 138]. The similar pattern is observed in the percoll gradient [137].

Therefore, the pre-synapses of all listed mediatory systems are present in the heavy fraction of synaptosomes with the exception of the opioid system, which synaptic transmission is absent in the caudal brainstem.

In accordance with the above literature, any of them dominate the heavy fraction or constitute at least half of the quantity/activity of the corresponding mediatory marker in the light fraction of synaptosomes. However, it is turned out in our case that the ratios between mediators within the light and heavy fractions are not as important when compared with their ratios within the heavy fraction. It is well known that glutamate and GABA are the prevailing neurotransmitters in any brain formation. This is manifested in synaptosomal fractions. For example, according to a comparative study of Johnson and Roberts [134], in the heavy fraction of the whole mouse brain, the content of glutamate-GABA-glycine-5-HT in the percentage distribution was 52-36-4-9%.

The relationships in the fraction between the protein content of the pre-synapses with different mediators would be similar. Therefore, the percentage of the sought-for mediator in the heavy fraction should be more than 18%, that is, above the fall in the protein content in our research. This requirement is consistent only with glutamate and GABA. According to the strictest calculation, they will dominate and be more than 33–23% in the heavy synaptosomal fraction when you consider that the entire brain glycine level is concentrated in the caudal brain stem [129] and that the heavy fraction also includes pre-synapses with some other “small “mediators (adenosinergic, cholinergic, etc.).

However, if GABA is the main inhibitory neurotransmitter of the brain, glutamate is the major excitatory neurotransmitter, including their physiological effects. Therefore, in apnoea, mechanisms may be involved only in a small part of a total pool of glutamate neurotransmission. Nevertheless, it is reasonable to assume that the pre-synapses of the powerful glutamatergic afferents in NTS are concentrated in the heavy fraction of synaptosomes by similarity with the pre-synapses of the cholinergic C-fibres, and thus, their concentration may be sufficiently representative in the heavy fraction of the caudal brainstem.

Taken together, it seems that GABAergic or glutamatergic neurons are the principal candidates in the hypoxic preconditioning mechanism of the reduction of the pre-synapses in the corresponding apnoeic sites of the caudal brainstem of the high-resistant rats. Perhaps HBH initiates signalling to pre-synapse reduction in the multiple mediatory neuronal populations (and in this case, the greatest interest represents the 5-HTergic system) but with the obligatory participation of GABAergic or glutamatergic neurons.

5.1.3. In the intact rats

In the intact rats, in the caudal brainstem (Figure 5c), HBH provoked an interconnected increase in the cChAT activity and c-protein content in the light synaptosomal fraction (r = +0.928, p < 0.02, n = 6) and a decrease in the mChAT activity and m-protein content (r = +0.933, p < 0.02, n = 6) in the heavy fraction. Changes in the activity of mChAT and content of m-protein in the heavy fraction were inversely proportional to the changes in cChAT activity and c-protein content in the light fraction (n = 6: mChAT-cChAT, r = −0.962, p < 0.02; m-protein–c-protein, r = −0.921, p < 0.05). Note that significant interfraction correlations between the light and heavy synaptosomal fractions were not found after HBH in the pre-testing rat groups.

We believe that in the intact rats, HBH initiated the transformation of cholinergic pre-synapses from the heavy fraction of synaptosomes, which altered their density characteristics, and during gradient fractionation, the transformed presynaptic population appeared in the light fraction. Moreover, cChAT activated in the transformed pre-synapses [112].

Activation of acetylcholine synthesis and non-quantum leakage in response to HBH points to the direct involvement of the relevant neurons in the preconditioning mechanisms in the intact caudal brainstem. Several respiratory-related sites exist in the VLM in which acetylcholine stimulated breathing and maintained an inspiration through mChR and/or nAChRs. Also, innervation of DFA by acetylcholine through nAChRs initiated the elevation of cerebral blood flow [108, 109].

In the intact rats, cChAT was activated in the cortical interneurons (Figure 6c, the heavy fraction of synaptosomes [124, 125]). There was no correlation between cChAT activity in the caudal brainstem and cortex in this rat group because of the absence of a direct link between the brain stem neurons and the cortical cholinergic interneurons.

Acetylcholine synthesis activation under HBH in the cortical interneurons could be related to their function of redistribution of the blood flow towards the brain. With respect to cerebral vessels, direct contacts with small cortical vessels and vasodilator effects of both the cholinergic projective neurons and interneurons were detected [139, 140, 141]. Thereby in intact brain, the cortical cholinergic interneurons might be involved in the local mechanisms to maintain the cerebral blood flow.

Thus, the intact rats had a synaptic response to HBH, the opposite of that of pre-tested rats: the activation of cardiorespiratory functions dominated in the intact rats, while the inhibition of pathways initiating apnoea appeared in the pre-tested rats. Apparently, the single pre-testing under SHBH altered synaptic and neuronal preconditioning mechanisms. The variety of neuronal pathways to achieve the same physiological effect demonstrates a great adaptive potential of brain. It seems, such adaptive possibilities are mortgaged by the composite, netted organisation of the respiratory centre. But it is not known whether all these mechanisms will go in the intact rats if theirs to activate, for example, pharmacologically.

In the total, HBH preconditioning eliminates the differences in resistance to SHBH between the intact, high- and low-resistant groups of rats with different innate resistance to severe hypoxia and prior hypoxic experiences. The same preconditioning effects of HBH in the intact rats and pre-tested under SHBH can be explained only by the fact that HBH preconditioning is realised by its own mechanisms, which do not depend on innate resistance to SHBH and prior hypoxic experiences.

At the same time, the resistance to SHBH initiated by HBH showed high rat-to-rat variability. So, the problem appeared to be the absence of methods for prediction of efficiency of hypoxic preconditioning.

Recently, such test was detected. It was a pre-pulse inhibition (PPI) estimated in the model of the acoustic startle reaction.


6. Experiments on the protocol number 2

It was found a correspondence between the values of PPI and T initiated by HBH, and the HBH efficiency was reliably and negatively correlated with PPI (Figure 7). The PPI in acoustic sensorimotor startle reaction is a well-known model that was developed in the second half of twentieth century for neurobiology, especially psychiatry [142], and these were the first direct experimental data to report the relationship between PPI and hypoxic preconditioning pathways (RF patent 2571603).

Figure 7.

The graph of dependence of the HBH preconditioning efficiency (T) on the rate of PPI. T, a time before apnoea. Grey marks, individual values of the rat resistance to SHBH after HBH. The significant negative correlation takes place between T and PPI values, Pearson’s r-criterion test.

In our recent publication [114] using literary data, we substantiated that acetylcholine, via nAChRs and especially via α7 nAChRs, is involved in hypoxic and ischaemic preconditioning and that an interconnection exists between α7 nAChRs, hypoxic preconditioning and PPI. Thereby in the pharmacological experiments, we investigated the effects of selective agonists of α4β4 and α7 nAChRs RJR and PNU, respectively, and PNU solvent DMSO on the HBH preconditioning. PPI measures were compared with the HBH-initiated preconditioning (resistance to SHBH) and with the effects of drugs on the HBH preconditioning efficiency (Figure 2).

RJR had no effect on the adaptive action of HBH. All the values of resistance in this group of rats ideally fitted into the variation series of T values of the HBH group (Figure 8).

Figure 8.

The influence on HBH preconditioning of the selective agonist α4β2 nAChRs metanicotine RJR 2304 (RJR). Grey marks, a time before apnoea after HBH as shown in Figure 7; light marks, a time before apnoea after RJR + HBH, Pearson’s r-criterion test.

Unlike RJR, PNU inversed the effects of HBH (Figure 9B), and it was especially clearly observed in the DMSO group (Figure 9C). Moreover, when the graphs of HBH and DMSO groups were combined, the interval of PPI = 0.36–0.40 (36–40%) was found (Figure 10). Above these values of PPI, DMSO potentiated the effects of HBH, and lower these values of PPI, DMSO, on the contrary, inhibited them. On the same PPI interval, the directionality of the action of PNU on DMSO effects was divided (Figure 11).

Figure 9.

The influence on HBH preconditioning of the selective agonist α7 nAChRs PNU-282,987 (PNU) and its solvent dimethyl sulfoxide (DMSO). Grey marks (A), T values in the HBH rat group as shown in Figure 7; black marks (B), T values in the PNU rat group; light marks (C), T values in the DMSO rat group. The significant negative correlation between PPI and T values after HBH inversed into the positive correlation under influence of PNU and significantly under DMSO influence, Pearson’s r-criterion test.

Figure 10.

Combined graphs of the dependence of the HBH preconditioning efficiency (T) on the rate of PPI in the HBH and DMSO groups. Grey marks, T values in the HBH rat group as shown in Figure 7; light marks, T values in the DMSO rat group. Vertical dotted lines indicate the values of PPI 0.36 and 0.40. The figures under the x-axis are given for orientation and denote the location of the corresponding values of PPI on the axis. The relationship between T values in the compared groups differs on the opposite sides of PPI = 0.36–0.40.

Figure 11.

Influence of DMSO and PNU on the HBH preconditioning efficiency in subgroups with PPI > 0.4 (A) and PPI < 0.4 (B). Values are expressed as means ± SE. Grey bars, HBH rat group; light bars, DMSO group; black bars, PNU group. *p < 0.05 and **p < 0.025 compared with T values in the relevant HBH groups, ##p < 0.025 compared with T values in the relevant DMSO group, Fisher’s exact test.

Analysis of the literature data revealed the following: (1) PNU in the low doses used (about 2 and 20 nM in the brain) had a desensitising effect on α7 nAChRs, that is, acted as an antagonist [143, 144]; (2) DMSO has the various biological activities, but in the low concentrations used (hundredths or thousandths of a per cent in the brain), it had only anticholinesterase action, that is, activating effect on the cholinergic system [145], and it was found that the anticholinesterase (neuroprotective) action is realised through the modulation of expression of nAChR genes that were shown for α7 and α4 subunits of nAChRs [146, 147, 148]. For details, see [114].

These data indicate the involvement of α7 nAChRs in the mechanisms of HBH preconditioning and explain the antagonism of PNU and DMSO actions. But the literature data do not explain the oppositely directed effects of DMSO and PNU on HBH preconditioning at the PPI boundary = 36–40%. Nevertheless, the existence of the interface does not seem random. Recently, it was found that the predisposed and resistant rats to convulsions in the hippocampal partial kindling model were differed in PPI: the resistant to convulsion rats had PPI of 36–58%, and the unstable to convulsion rats had in all experiments PPI larger, which was selectively susceptible to pronounced variability [149].

Also, key brain structures involved in the innate mechanisms of hypoxic preconditioning are unknown. We suppose the obligatory participation of caudal brainstem. In studies related to PPI, the key structure is the hippocampus [149, 150, 151, 152]. In our study, in the intact rat group with not arranged related to PPI, no reaction was shown in the hippocampus. We hope to clarify this problem somewhat in the planned neurochemical studies of synaptic pool in the intact rats pre-tested with PPI.


7. Conclusion

  1. A search study of the effects and neuronal mechanisms of hypoxic preconditioning were carried out, and the certain results in this direction were obtained using the HBH model.

  2. It has been revealed that the model of acoustic start-reaction can be used to predict the efficiency of hypoxic preconditioning and the study of their innate mechanisms because the magnitude of criterion for this model of PPI has the reverse dependence related to the HBH-initiated resistance to SHBH in the intact rats.

  3. The pre-testing of intact rats at the PPI revealed the presence of oppositely directed cholinergic mechanisms of hypoxic preconditioning, separated at the border of PPI = 36–40%, and the α7 nAChRs participation in both the mechanisms.



We are grateful to the direction of Institute of General Pathology and Pathophysiology that supported and funded our research and to the direction of P.K. Anokhin’ Institute of Normal Physiology for the successful cooperation. And we would like to thank the team of the Proof-Reading-Service for editing and proofreading the English of our chapter.


  1. 1. Hawaleshka A, Jacobsohn E. Ischaemic preconditioning: Mechanisms and potential clinical applications. Canadian Journal of Anesthesia. 1998;45:670-682. DOI: 10.1007/BF03012100
  2. 2. Lukyanova LD, Germanova EL, Tsibina TA, Kopaladze RA, Dudchenko A. Efficiency and mechanism for different regimens of hypoxic training. The possibility of optimization of hypoxic therapy. Patogenez. 2008;6:32-36 (In Russian)
  3. 3. Park HK, Seol IJ, Kim KS. Protective effect of hypoxic preconditioning on hypoxic-ischaemic injured newborn rats. Journal of Korean Medical Science. 2011;26:1495-1500. DOI: 10.3346/jkms.2011.26.11.1495
  4. 4. Sazontova TG, Bolotova AV, Bedareva IV, Kostina NV, Yurasov AR, Arkhipenko YV. Hypoxia-inducible factor (HIF-1a), HSPs, antioxidant enzymes and membrane resistance to ROS in endurance exercise performance after adaptive hypoxic preconditioning. In: Wang P, Kuo C-H, Takeda N, Singal PK, editors. Adaptation Biology and Medicine. New Delhi: Narosa Publishing House; 2011. pp. 161-179
  5. 5. Samoilov MO, Rybnikova EA, Churilova AV. Signal molecular and hormonal mechanisms of formation of the hypoxic preconditioning protective effects. Patologicheskaia Fiziologiia i Eksperimental’naia Terapiia. 2012;56:3-10 (In Russian)
  6. 6. Das M, Das DK. Molecular mechanism of preconditioning. IUBMB Life. 2008;60:199-203.
  7. 7. Kirova II. Effect of hypoxia on dynamics of HIF-1alpha level in the cerebral cortex and development of adaptation in rats with different resistance to hypoxia. Patologicheskaia Fiziologiia i Èksperimental'naia Terapiia. 2012;56:51-55 (In Russian)
  8. 8. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischaemia: A delay of lethal cell injury in ischaemic myocardium. Circulation. 1986;74:1124-1136
  9. 9. Richard V, Murry CE, Reimer KA. Healing of myocardial infarcts in dogs. Effects of late reperfusion. Circulation. 1995;92:1891-1901
  10. 10. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, Kamada T. “Ischaemic tolerance” phenomenon found in the brain. Brain Research. 1990;528:21-24. DOI: 10.1016/0006-8993(90)90189-I
  11. 11. Gidday JM, Fitzgibbons JC, Shah AR, Park TS. Neuroprotection from ischaemic brain injury by hypoxic preconditioning in the neonatal rat. Neuroscience Letters. 1994;168:221-224. DOI: 10.1016/0304-3940(94)90455-3
  12. 12. Samoilov MO. Brain and Adaptation. Molecular-Cellular Mechanisms. St. Petersburg: Turusel; 1999. 271 p. (In Russian)
  13. 13. Stone TW. Pre-conditioning protection in the brain. British Journal of Pharmacology. 2003;140:229-230. DOI: 10.1038/sj.bjp.0705441
  14. 14. Liu Y, Chen L, Xu X, Vicaut E, Sercombe R. Both ischaemic preconditioning and ghrelin administration protect hippocampus from ischaemia/reperfusion and upregulate uncoupling protein-2. BMC Physiology. 2009;9:17. DOI:10.1186/1472-6793-9-17
  15. 15. Speer R, Ratan RR. Hypoxic adaptation in the nervous system: Promise for novel therapeutics for acute and chronic neurodegeneration. Advances in Experimental Medicine and Biology. 2016;903:221-243. DOI: 10.1007/978-1-4899-7678-9_16
  16. 16. Tajima M, Katayose D, Bessho M, Isoyama S. Acute ischaemic preconditioning and chronic hypoxia independently increase myocardial tolerance to ischaemia. Cardiovascular Research. 1994;28:312-319. DOI: 10.1093/cvr/28.3.312
  17. 17. Duan Z, Zhang L, Liu J, Xiang X, Lin H. Early protective effect of total hypoxic preconditioning on rats against systemic injury from hemorrhagic shock and resuscitation. Journal of Surgical Research. 2012;178:842-850. DOI: 10.1016/j.jss.2012.04.069
  18. 18. Xu R, Sun Y, Chen Z, Yao Y, Ma G. Hypoxic preconditioning inhibits hypoxia-induced apoptosis of cardiac progenitor cells via the PI3K/Akt-DNMT1-p53 pathway. Scientific Reports. 2016;6:30922. DOI: 10.1038/srep30922
  19. 19. Lai IR, Chang KJ, Chen CF, Tsai HW. Transient limb ischaemia induces remote preconditioning in liver among rats: The protective role of heme oxygenase-1. Transplantation. 2006;81:1311-1317. DOI: 10.1097/
  20. 20. Chouker A, Ohta A, Martignoni A, Lukashev D, Zacharia LC, Jackson EK, Schnermann J, Ward JM, Kaufmann I, Klaunberg B, Sitkovsky MV, Thiel M. In vivo hypoxic preconditioning protects from warm liver ischaemia-reperfusion injury through the adenosine A2B receptor. Transplantation. 2012;94:894-902. DOI: 10.1097/TP.0b013e31826a9a46
  21. 21. Zhuonan Z, Sen G, Zhipeng J, Maoyou Z, Linglan Y, Gangping W, Cheng J, Zhongliang M, Tian J, Peijian Z, Kesen X. Hypoxia preconditioning induced HIF-1α promotes glucose metabolism and protects mitochondria in liver I/R injury. Clinics and Research in Hepatology and Gastroenterology. 2015;39:610-619. DOI: 10.1016/j.clinre.2014.12.012
  22. 22. Bernhardt WM, Campean V, Kany S, Jurgensen JS, Weidemann A, Warnecke C, Arend M, Klaus S, Gunzler V, Amann K, Willam C, Wiesener MS, Eckardt KU. Preconditional activation of hypoxia-inducible factors ameliorates ischaemic acute renal failure. Journal of the American Society of Nephrology. 2006;17:1970-1978. DOI: 10.1681/ASN.2005121302
  23. 23. Overath JM, Gauer S, Obermuller N, Schubert R, Schafer R, Geiger H, Baer PC. Short-term preconditioning enhances the therapeutic potential of adipose-derived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury. Experimental Cell Research. 2016;342:175-183. DOI: 10.1016/j.yexcr.2016.03.002
  24. 24. Saether K, Hilaire G, Monteau R. Dorsal and ventral respiratory groups of neurons in the medulla of the rat. Brain Research. 1987;419:87-96
  25. 25. Tarakanov IA, Safonov VA. Neurohumoral concept of central respiratory regulation. Patogenez. 2003;1:11-24 (In Russian)
  26. 26. Richter DW, Smith JC. Respiratory rhythm generation in vivo. Physiology (Bethesda). 2014;29:58-71
  27. 27. Loeschcke HH. Central chemosensitivity and the reaction theory. The Journal of Physiology. 1982;332:1-24
  28. 28. Reis DJ, Ruggiero DA, Morrison SF. The C1 area of the rostral ventrolateral medulla oblongata: A critical brainstem region for control of resting and reflex integration of arterial pressure. American Journal of Hypertension. 1989;2:363S-374S
  29. 29. Richter DW, Bischoff A, Anders KM, Windhorst U. Response of the medullary respiratory network of the cat to hypoxia. The Journal of Physiology. 1991;443:231-256
  30. 30. Sun MK, Reis DJ. Central neural mechanisms mediating excitation of sympathetic neurons by hypoxia. Progress in Neurobiology. 1994;44:197-219
  31. 31. Thomas T, Marshall JM. Interdependence of respiratory and cardiovascular changes induced by systemic hypoxia in the rat: The roles of adenosine. The Journal of Physiology. 1994;480:627-636
  32. 32. Richter DW, Schmidt-Garcon P, Pierrefiche O, Bischoff AM, Lalley PM. Neurotransmitters and neuromodulators controlling the hypoxic respiratory response in anaesthetized cats. The Journal of Physiology. 1999;514:567-578
  33. 33. Nakai M, Tamaki K, Ogata J, Matsui Y, Maeda M. Parasympathetic cerebrovasodilator center of the facial nerve. Circulation Research. 1993;72:470-475
  34. 34. Golanov EV, Reis DJ. Contribution of oxygen-sensitive neurons of the rostral ventrolateral medulla to hypoxic cerebral vasodilatation in the rat. The Journal of Physiology. 1996;495:201-216
  35. 35. Reis DJ, Golanov EV, Galea E, Feinstein DL. Central neurogenic neuroprotection: Central neural systems that protect the brain from hypoxia and ischaemia. Annals of the New York Academy of Sciences. 1997;835:168-186
  36. 36. Sanotskaya NV, Matsievskii DD, Lebedeva MA. Acute hypoxia influence on pulmonary and systemic blood circulation. Patogenez. 2012;10:56-59 (In Russian)
  37. 37. Gong CL, Leung YM, Huang YP, Lin NN, Hung YW, et al. Nicotine activation of neuronal nitric oxide synthase and guanylyl cyclase in the medulla increases blood flow of the common carotid artery in cats. Neuroscience Letters. 2010;486:122-126
  38. 38. Gong CL, Leung YM, Wang MR, Lin NN, Lee TJ, et al. Neurochemicals involved in medullary control of common carotid blood flow. Neuropharmacology. 2013;11:513-520
  39. 39. Golanov EV, Ruggiero DA, Reis DJ. A brainstem area mediating cerebrovascular and EEG responses to hypoxic excitation of rostral ventrolateral medulla in rat. The Journal of Physiology. 2000;529:413-429
  40. 40. Sanotskaia NV, Matsievskiĭ DD, Tarakanov IA. Changes in hemodynamics and respiration in animals with various resistance to acute hypoxia. Biulleten' Eksperimental'noĭ Biologii i Meditsiny. 1999;128:286-290 (In Russian)
  41. 41. Sanotskaya NV, Matsievskii DD, Lebedeva MA. Changes in hemodynamics and respiration in rats with different resistance to acute hypoxia. Bulletin of Experimental Biology and Medicine. 2004;138:18-22
  42. 42. Sanotskaya NV, Matsievskii DD, Lebedeva MA. Effect of picrotoxin on organism's resistance to acute severe hypoxia. Bulletin of Experimental Biology and Medicine. 2008;145:177-180
  43. 43. Tabata M, Kurosawa H, Kikuchi Y, Hida W, Ogawa H, Okabe S, Tun Y, Hattori T, Shirato K. Role of GABA within the nucleus tractus solitarii in the hypoxic ventilatory decline of awake rats. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2001;281:R1411-R1419
  44. 44. Xing T, Fong AY, Bautista TG, Pilowsky PM. Acute intermittent hypoxia induced neural plasticity in respiratory motor control. Clinical and Experimental Pharmacology & Physiology. 2013;40:602-609
  45. 45. Belchenko LA. Adaptation of humans and animals to various kinds of hypoxia. Soros Educational Journal. 2001;7:1-7 (In Russian)
  46. 46. Chizhov AYa, Bludov AA. Mechanisms and foundations of resonant hypoxitherapy. In: Luk’yanova LD, Ushakov IB, editors. Problems of Hypoxia: Molecular, Physiological, and Medical Aspects. Voronezh: Istoki; 2004. pp. 519-568 (In Russian)
  47. 47. Araki T, Kato H, Fujiwara T, Kogure K, Itoyama Y. Alteration of [3H]hemicholinium-3 binding in the post-ischaemic gerbil brain. Neuroreport. 1995;6:561-564
  48. 48. Mitchell RA, Berger AJ. Neural regulation of respiration. The American Review of Respiratory Disease. 1975;111:206-224
  49. 49. Edlow BL, McNab JA, Witzel T, Kinney HC. The structural connectome of the human central homeostatic network. Brain Connectivity. 2016;6:187-200
  50. 50. Tarakanov IA, Tikhomirova LN, Safonov VA. Effect of opioids on mechanoreflexive respiration control. Bulletin of Experimental Biology and Medicine. 2005;139:388-390
  51. 51. Manzke T, Niebert M, Koch UR, Caley A, Vogelgesang S, Hülsmann S, Ponimaskin E, Müller U, Smart TG, Harvey RJ, Richter DW. Serotonin receptor 1A-modulated phosphorylation of glycine receptor α3 controls breathing in mice. The Journal of Clinical Investigation. 2010;120:4118-4128
  52. 52. Montandon G, Horner R. CrossTalk proposal: The preBötzinger complex is essential for the respiratory depression following systemic administration of opioid analgesics. The Journal of Physiology. 2014;592:1159-1162
  53. 53. Lalley PM, Pilowsky PM, Forster HV, Zuperku EJ. CrossTalk opposing view: The pre-Botzinger complex is not essential for respiratory depression following systemic administration of opioid analgesics. The Journal of Physiology. 2014;592:1163-1166
  54. 54. Tikhomirova LN, Safina NF, Tarakanov IA. The role of opioidergic and GABAergic systems in the mechanosensitivity regulation of the respiratory system in rats. Patologicheskaia Fiziologiia i Èksperimental'naia Terapiia. 2015;59:26-29 (In Russian)
  55. 55. Pires JG, Silva SR, Ramage AG, Futuro-Neto HA. Evidence that 5-HT3 receptors in the nucleus tractus solitarius and other brainstem areas modulate the vagal bradycardia evoked by activation of the von Bezold-Jarisch reflex in the anesthetized rat. Brain Research. 1998;791:229-234
  56. 56. Lalley PM, Benacka R, Bischoff AM, Richter DW. Nucleus raphe obscurus evokes 5-HT-1A receptor-mediated modulation of respiratory neurons. Brain Research. 1997;747:156-159
  57. 57. Verner TA, Goodchild AK, Pilowsky PM. A mapping study of cardiorespiratory responses to chemical stimulation of the midline medulla oblongata in ventilated and freely breathing rats. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2004;287:R411-R421
  58. 58. Verner TA, Pilowsky PM, Goodchild AK. Retrograde projections to a discrete apneic site in the midline medulla oblongata of the rat. Brain Research. 2008;1208:28-36
  59. 59. Mirzoian RS, Romanycheva NA, Gan'shina TS, Aleksandrin VV, Volobueva TI, Aleksandrov PN. The nonuniform sensitivity of the cerebral vessels to serotonin. Eksperimental'naia i Klinicheskaia Farmakologiia. 1993;56:22-25 (In Russian)
  60. 60. Mirzoian RS, Topchian AV, Gan'shina TS, Kostochka LM. Tropoxin and cerebrovascular effects of serotonin. Eksperimental'naia i Klinicheskaia Farmakologiia. 2000;63:21-23 (In Russian)
  61. 61. Aleksandrin VV, Tarasova NN, Tarakanov IA. Effect of serotonin on respiration, cerebral circulation, and blood pressure in rats. Bulletin of Experimental Biology and Medicine. 2005;139:64-67
  62. 62. Baccini G, Mlinar B, Audero E, Gross CT, Corradetti R. Impaired chemosensitivity of mouse dorsal raphe serotonergic neurons overexpressing serotonin 1A (Htr1a) receptors. PLoS One. 2012;7:e45072. DOI:10.1371/journal.pone.0045072
  63. 63. Sapru HN. Carotid chemoreflex. Neural pathways and transmitters. Advances in Experimental Medicine and Biology. 1996;410:357-364
  64. 64. Aicher SA, Milner TA, Pickel VM, Reis DJ. Anatomical substrates for baroreflex sympathoinhibition in the rat. Brain Research Bulletin. 2000;51:107-110
  65. 65. Seifert E, Trippenbach T. Baclofen attenuates cardiorespiratory effects of vagal C fiber stimulation in rats. Canadian Journal of Physiology and Pharmacology. 1995;73:1485-1494
  66. 66. Subramanian HH, Chow CM, Balnave RJ. Identification of different types of respiratory neurones in the dorsal brainstem nucleus tractus solitarius of the rat. Brain Research. 2007;1141:119-132
  67. 67. Tarakanov IA, Tarasova NN, Safronov VA. Effects of lithium hydroxybutyrate and vagotomy on respiratory arrest caused by systemic serotonin administration in rats. Eksperimental'naia i Klinicheskaia Farmakologiia. 2005;68:30-35 (In Russian)
  68. 68. Tarakanov IA, Tarasova NN, Belova EA, Safonov VA. Effect of phenibut on the respiratory arrest caused by serotonin. Eksperimental'naia i Klinicheskaia Farmakologiia. 2006;69:28-32 (In Russian)
  69. 69. Kryzhanovskiĭ GN, Tarakanov IA, Safonov VA. The participation of the GABAergic system of the brain in shaping the breathing rhythm. Fiziologicheskiĭ Zhurnal Imeni I.M. Sechenova. 1993;79:13-23 (In Russian)
  70. 70. Suzuki M, Nishina M, Nakamura S, Maruyama K. Endogenous GABA in the commissural subnucleus of the NTS inhibits the carotid chemoreceptor reflex via GABA A receptors in rats. Journal of Neural Transmission (Vienna). 2003;110:211-218
  71. 71. Schmid K, Foutz AS, Denavit-Saubié M. Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons. Brain Research. 1996;710:150-160
  72. 72. Vediasova OA, Man'shina NG, Safonov VA, Tarakanov IA. Respiratory reactions on microinjections of GABA and baclofen into the Bötzinger complex and the pre-Bötzinger complex in rats. Rossiĭskii Fiziologicheskiĭ Zhurnal Imeni I.M. Sechenova. 2012;98:618-626 (In Russian)
  73. 73. Bongianni F, Mutolo D, Cinelli E, Pantaleo T. Respiratory responses induced by blockades of GABA and glycine receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit. Brain Research. 2010;1344:134-147
  74. 74. Pierrefiche O, Schwarzacher SW, Bischoff AM, Richter DW. Blockade of synaptic inhibition within the pre-Bötzinger complex in the cat suppresses respiratory rhythm generation in vivo. The Journal of Physiology. 1998;509:245-254
  75. 75. Smith JC, Abdala AP, Koizumi H, Rybak IA, Paton JF. Spatial and functional architecture of the mammalian brain stem respiratory network: A hierarchy of three oscillatory mechanisms. Journal of Neurophysiology. 2007;98:3370-3387
  76. 76. Ezure K, Tanaka I, Miyazaki M. Electrophysiological and pharmacological analysis of synaptic inputs to pulmonary rapidly adapting receptor relay neurons in the rat. Experimental Brain Research. 1999;128:471-480
  77. 77. Heesch CM, Laiprasert JD, Kvochina L. RVLM glycine receptors mediate GABAA and GABAB independent sympathoinhibition from CVLM in rats. Brain Research. 2006;1125:46-59
  78. 78. Talman WT, Robertson SC. Glycine, like glutamate, microinjected into the nucleus tractus solitarii of rat decreases arterial pressure and heart rate. Brain Research. 1989;47:7-13
  79. 79. Mauad H, Colombari E, Bonagamba LG, Machado BH. Glycine blocks the pressor response to L-glutamate microinjected into the nucleus tractus solitarii of conscious rats. Brazilian Journal of Medical and Biological Research. 1995;28:699-704
  80. 80. Thomas T, Spyer KM. The role of adenosine receptors in the rostral ventrolateral medulla in the cardiovascular response to defence area stimulation in the rat. Experimental Physiology. 1996;81:67-77
  81. 81. Thomas T, St Lambert JH, Dashwood MR, Spyer KM. Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control. Neuroscience. 2000;95:513-518
  82. 82. Minic Z, O'Leary DS, Scislo TJ. Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs. Autonomic Neuroscience. 2014;180:32-42
  83. 83. Pedata F, Dettori I, Coppi E, Melani A, Fusco I, Corradetti R, Pugliese AM. Purinergic signalling in brain ischaemia. Neuropharmacology. 2015;104:105-130
  84. 84. Pugliese AM, Coppi E, Volpini R, Cristalli G, Corradetti R, Jeong LS, Jacobson KA, Pedata F. Role of adenosine A3 receptors on CA1 hippocampal neurotransmission during oxygen-glucose deprivation episodes of different duration. Biochemical Pharmacology. 2007;74:768-779
  85. 85. St Lambert JH, Dashwood MR, Spyer KM. Role of brainstem adenosine A1 receptors in the cardiovascular response to hypothalamic defence area stimulation in the anaesthetized rat. British Journal of Pharmacology. 1996;117:277-282
  86. 86. Gutierres JM, Carvalho FB, Schetinger MR, Rodrigues MV, Schmatz R, Pimentel VC, Vieira JM, Rosa MM, Marisco P, Ribeiro DA, Leal C, Rubin MA, Mazzanti CM, Spanevello R. Protective effects of anthocyanins on the edtonucleotidase activity in the impairment of memory induced by scopolamine in adult rats. Life Sciences. 2012;91:1221-1228
  87. 87. Corti F, Cellai L, Melani A, Donati C, Bruni P, Pedata F. Adenosine is present in rat brain synaptic vesicles. Neuroreport. 2013;24:982-987
  88. 88. Lawrence AJ, Castillo-Meléndez M, Jarrott B. [3H]adenosine transport in rat dorsal brain stem using a crude synaptosomal preparation. Neurochemistry International. 1994;25:221-226
  89. 89. Castillo-Meléndez M, Jarrott B, Lawrence AJ. Markers of adenosine removal in normotensive and hypertensive rat nervous tissue. Hypertension. 1996;28:1026-1033
  90. 90. Barraco RA, El-Ridi MR, Ergene E, Phillis JW. Adenosine receptor subtypes in the brainstem mediate distinct cardiovascular response patterns. Brain Research Bulletin. 1991;26:59-84
  91. 91. Mosqueda-Garcia R, Tseng CJ, Appalsamy M, Beck C, Robertson D. Cardiovascular excitatory effects of adenosine in the nucleus of the solitary tract. Hypertension. 1991;18:494-502
  92. 92. Scislo TJ, O'Leary DS. Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors. American Journal of Physiology. Heart and Circulatory Physiology. 2002;283:H1588-H1599
  93. 93. White PJ, Rose'Meyer RB, Hope W. Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat. British Journal of Pharmacology. 1996;117:305-308
  94. 94. Minic Z, O'Leary DS, Scislo TJ. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism. American Journal of Physiology. Heart and Circulatory Physiology. 2015;309:H185-H197
  95. 95. Schmidt C, Bellingham MC, Richter DW. Adenosinergic modulation of respiratory neurons and hypoxic responses in the anaesthetized cat. The Journal of Physiology. 1995;483:769-781
  96. 96. Neylon M, Marshall JM. The role of adenosine in the respiratory and cardiovascular response to systemic hypoxia in the rat. The Journal of Physiology. 1991;440:529-545
  97. 97. Haxhiu MA, Kc P, Moore CT, Acquah SS, Wilson CG, Zaidi SI, Massari VJ, Ferguson DG. Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses. Journal of Applied Physiology. 2005;98:1961-1982
  98. 98. Feldberg W, Guertzenstein PG. A vasodepressor effect of pentobarbitone sodium. The Journal of Physiology. 1972;224:83-103
  99. 99. Ruggiero DA, Giuliano R, Anwar M, Stornetta R, Reis DJ. Anatomical substrates of cholinergic-autonomic regulation in the rat. The Journal of Comparative Neurology. 1990;292:1-53
  100. 100. Rye DB, Lee HJ, Saper CB, Wainer BH. Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum in the rat. The Journal of Comparative Neurology. 1988;269:315-341
  101. 101. Woolf NJ, Butcher LL. Cholinergic systems in the rat brain: IV. Descending projections of the pontomesencephalic tegmentum. Brain Research Bulletin. 1989;23:519-540
  102. 102. Jones BE. Immunohistochemical study of choline acetyltransferase immunoreactive processes and cells innervating the pontomedullary reticular formation in the rat. The Journal of Comparative Neurology. 1990;295:485-514
  103. 103. Woolf NJ. Cholinergic systems in mammalian brain and spinal cord. Progress in Neurobiology. 1991;37:475-524
  104. 104. Reynolds DJ, Lowenstein PR, Moorman JM, Grahame-Smith DG, Leslie RA. Evidence for cholinergic vagal afferents and vagal presynaptic M1 receptors in the ferret. Neurochemistry International. 1994;25:455-464
  105. 105. Schäfer MK, Eiden LE, Weihe E. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. I. Central nervous system. Neuroscience. 1998;84:331-359
  106. 106. Armstrong DM, Rotler A, Hersh LB, Pickel VM. Localization of choline acetyltransferase in perikarya and dendrites within the nuclei of the solitary tracts. Journal of Neuroscience Research. 1988;20:279-290
  107. 107. Kc P, Mayer CA, Haxhiu MA. Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: The absence of non-cholinergic neurons. Journal of Applied Physiology. 2004;97:1508-1517
  108. 108. Zakharova EI, Dudchenko AM. Variety of neuronal pathways to achieve the same hypoxic preconditioning effect. Biochemistry & Physiology. 2016;5:4. DOI: 10.4172/2168-9652.1000212
  109. 109. Zakharova EI, Dudchenko AM. Variety of neuronal pathways to achieve the same hypoxic preconditioning effect. In: Top 10 Contributions on Biochemistry. 2nd ed. Avid Science; 2018.ch02 (In press)
  110. 110. Fonnum F. Radiochemical microassays for the determination of choline acetyltransferase and acetylcholinesterase activities. The Biochemical Journal. 1969;115:465-472
  111. 111. Lowry OH, Rosenbrough NJ, Farr AL, Randall R. Protein measurement with Folin phenol reagent. The Journal of Biological Chemistry. 1959;19:265-275
  112. 112. Zakharova EI, Germanova EL, Kopaladze RA, Dudchenko AM. Central cholinergic systems in the mechanisms of hypoxic preconditioning: Diverse pathways of synaptic reorganization in vivo. Neurochemical Journal. 2013;7:45-55
  113. 113. Zakharova EI, Dudchenko AM. Hypoxic preconditioning eliminates differences in the innate resistance of rats to severe hypoxia. Journal of Biomedical Science and Engineering. 2016;9:563-575. DOI: 10.4236/jbise.2016.912049
  114. 114. Zakharova EI, Storozheva ZI, Proshin AT, Monakov MYu, Dudchenko AM. The prepulse inhibition of startle (PPI) predicts an efficiency of hypoxic preconditioning. Journal of Biomedical Science and Engineering. 2018;11(1):10-25
  115. 115. Purshottam T, Ghosh NC. Effect of acetazolamide (diamox) at different dose levels on survival time of rats under acute hypoxia and on Na+-K+-ATP-ase activity of rat tissue microsomes. Aerospace Medicine. 1972;43:610-613
  116. 116. Dudchenko AM. Comparison of mitochondrial dehydrogenase of cerebral cortex in rats, having different sensitivity to hypoxia. In: Severin SE, editor. Mitochondria. Transport of Electrons and Energy Conversion. Moscow: Nauka; 1976. pp. 177-182 (In Russian)
  117. 117. Berezovski VA, Boiko KA, Klimenko KS, Levchenko MN, Nazarenko AI, Shumitskaya NM. Hypoxia and Individual Features of Reactivity. Kiev: Naukova Dumka; 1978 (In Russian)
  118. 118. Luk’ianova LD. Bioenergetic hypoxia: Definition, mechanisms, and methods of correction. Bulletin of Experimental Biology and Medicine. 1997;24:835-843. 10.1007/BF02446979
  119. 119. Zakharova EI, Dudchenko AM, Germanova EL. Effects of preconditioning on the resistance to acute hypobaric hypoxia and their correction with selective antagonists of nicotinic receptors. Bulletin of Experimental Biology and Medicine. 2011;151:179-182. DOI: 10.1007/s10517-011-1283-2
  120. 120. Pevtsov EF, Storozheva ZI, Proshin AT, Pevtsova EI. A hardware-and-software system for experimental studies of the acoustic startle response in laboratory rodents. Bulletin of Experimental Biology and Medicine. 2016;160:410-413. DOI: 10.1007/s10517-016-3183-y
  121. 121. Undem BJ, Chuaychoo B, Lee MG, Weinreich D, Myers AC, et al. Subtypes of vagal afferent C-fibres in guinea-pig lungs. The Journal of Physiology. 2004;556:905-917
  122. 122. Filippova LV, Nozdrachev AD. An overview of the pulmonary sensory receptors. Uspekhi Fiziologicheskikh Nauk. 2013;44:93-112 (In Russian)
  123. 123. Bonham AC, Joad JP. Neurones in commissural nucleus tractus solitarii required for full expression of the pulmonary C fibre reflex in rat. The Journal of Physiology. 1991;441:95-112
  124. 124. Zakharova EI, Dudchenko AM, Svinov MM, Ivanov DS, Ignat’ev IV. Comparative characteristic of the brain cholinergic systems in rats with low and high resistance to oxygen deficiency. Neirokhimiya. 2001;18:119-131 (In Russian)
  125. 125. Zakharova EI, Dudchenko AM. Synaptic soluble and membrane-bound choline acetyltransferase as a marker of cholinergic function in vitro and in vivo. In: Heinbockel T, editor. Neurochemistry. Rijeka: InTech; 2014. p. 143-178. DOI: 10.5772/57074.ch05
  126. 126. Semba K, Reiner PB, McGeer EG, Fibiger HC. Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry and electrophysiology in the rat. The Journal of Comparative Neurology. 1988;267:433-453
  127. 127. Kuhar MJ, Green AI, Snyder SH, Gfeller E. Separation of synaptosomes storing catecholamines and gamma-aminobutyric acid in rat corpus striatum. Brain Research. 1970;21:405-417
  128. 128. Gfeller E, Kuhar MJ, Snyder SH. Neurotransmitter-specific synaptosomes in rat corpus striatum: Morphological variations. Proceedings of the National Academy of Sciences of the United States of America. 1971;68:155-159
  129. 129. Arregui A, Logan WJ, Bennett JP, Snyder SH. Specific glycine—Accumulating synaptosomes in the spinal cord of rats. Proceedings of the National Academy of Sciences of the United States of America. 1972;69:3485-3489
  130. 130. Rodríguez de Lores G, De Robertis E. 5-Hydroxytryptophan decarboxylase activity in nerve engings of the rat brain. Journal of Neurochemistry. 1964;11:213-219
  131. 131. Wenk M, Von Hahn HP, Honegger CG. Partial separation of synaptosomes accumulating 4-aminobutyrate or glutamate by zonal centrifugation on a discontinuous sucrose gradient. Hoppe-Seyler's Zeitschrift für Physiologische Chemie. 1976;357:1469-1476
  132. 132. Kunert E, Dovedova EL. Effect of light deprivation on GABA metabolism in subcellular fractions of the rabbit visual system. Voprosy Meditsinskoĭ Khimii. 1979;25:460-466 (In Russian)
  133. 133. Uzbekov MG. Effect of light deprivation on binding activity of serotonin with light and heavy synaptosomes from various brain formations. Neuroscience and Behavioral Physiology. 1980;10:33-35
  134. 134. Johnson JL, Roberts E. Proline, glutamate and glutamine metabolism in mouse brain synaptosomes. Brain Research. 1984;323:247-256
  135. 135. Agaev TM, Kurbanova GA. The distribution of aspartate aminotransferase activity in the structures of the visual analyzer in the canine brain during postnatal ontogeny. Zhurnal Evoliutsionnoĭ Biokhimii i Fiziologii. 1994;30:192-197 (In Russian)
  136. 136. Dunkley PR, Jarvie PE, Heath JW, Kidd GJ, Rostas JA. A rapid method for isolation of synaptosomes on Percoll gradients. Brain Research. 1986;372:115-129
  137. 137. Dunkley PR, Heath JW, Harrison SM, Jarvie PE, Glenfield PJ, Rostas JA. A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: Homogeneity and morphology of subcellular fractions. Brain Research. 1988;441:59-71
  138. 138. Lathia D, Wesemann W. Serotonin uptake and release by biochemically characterized nerve endings isolated from rat brain by concomitant flotation and sedimentation centrifugation. Journal of Neural Transmission. 1975;37:111-126
  139. 139. Dauphin F, Lacombe P, Sercombe R, Hamel E, Seylaz J. Hypercapnia and stimulation of the substantia innominata increase rat frontal cortical blood flow by different cholinergic mechanisms. Brain Research. 1991;553:75-83
  140. 140. Chédotal A, Cozzari C, Faure MP, Hartman BK, Hamel E. Distinct choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) bipolar neurons project to local blood vessels in the rat cerebral cortex. Brain Research. 1994;646:181-193
  141. 141. Vaucher E, Hamel E. Cholinergic basal forebrain neurons project to cortical microvessels in the rat: Electron microscopic study with anterogradely transported Phaseolus vulgaris leucoagglutinin and choline acetyltransferase immunocytochemistry. The Journal of Neuroscience. 1995;15:7427-7441
  142. 142. Swerdlow NR, Braff DL, Geyer MA. Sensorimotor gating of the startle reflex: What we said 2 years ago, what has happened since then, and what comes next. Journal of Sychopharmacology. 2016;30:1072-1081. DOI: 10.1177/0269881116661075
  143. 143. Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX. Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. The European Journal of Neuroscience. 1997;9:2734-2742
  144. 144. Hajos M, Hurst RS, Hoffmann WE, Krause M, Wall TM, Higdon NR, Groppi VE. The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl] 4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats. Journal of Pharmacology and Experimental Therapeutics. 2005;312:1213-1222. DOI: 10.1124/jpet.104.076968
  145. 145. Sams Jr WM, Carroll NV. Cholinesterase inhibitory property of dimethyl sulphoxide. Nature. 1966;212:405. DOI: 10.1038/212405a0
  146. 146. Takada Y, Yonezawa A, Kume T, Katsuki H, Kaneko S, Sugimoto H, Akaike A. Nicotinic acetylcholine receptor-mediated neuroprotection by donepezil against glutamate neurotoxicity in rat cortical neurons. Journal of Pharmacology and Experimental Therapeutics. 2003;306:772-777. DOI: 10.1124/jpet.103.050104
  147. 147. Fujiki M, Kubo T, Kamida T, Sugita K, Hikawa T, Abe T, Ishii K, Kobayashi H. Neuroprotective and antiamnesic effect of donepezil, a nicotinic acetylcholine-receptor activator, on rats with concussive mild traumatic brain injury. Journal of Clinical Neuroscience. 2008;15:791-796. DOI: 10.1016/j.jocn.2007.07.002
  148. 148. Takada-Takatori Y, Kume T, Izumi Y, Ohgi Y, Niidome T, Fujii T, Sugimoto H, Akaike A. Roles of nicotinic receptors in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation. Biological & Pharmaceutical Bulletin. 2009;32:318-324. DOI: 10.1248/bpb.32.318
  149. 149. Ma J, Leung LS. Effects of hippocampal partial kindling on sensory and sensorimotor gating and methamphetamine-induced locomotion in kindling-prone and kindling-resistant rats. Epilepsy & Behavior. 2016;58:119-126. DOI: 10.1016/j.yebeh.2016.03.001
  150. 150. Ma J, Shen B, Rajakumar N, Leung LS. The medial septum mediates impairment of prepulse inhibition of acoustic startle induced by a hippocampal seizure or phencyclidine. Behavioural Brain Research. 2004;155(1):153-166
  151. 151. Cilia J, Cluderay JE, Robbins MJ, Reavill C, Southam E, Kew JN, Jones DN. Reversal of isolation-rearing-induced PPI deficits by an alpha7 nicotinic receptor agonist. Psychopharmacology. Oct 2005;182(2):214-219
  152. 152. McGarrity S, Mason R, Fone KC, Pezze M, Bast. Hippocampal neural disinhibition causes attentional and memory deficits. Cerebral Cortex. 2017;27(9):4447-4462. DOI: 10.1093/cercor/bhw247

Written By

Elena I. Zakharova, Zanaida I. Storozheva, Andrew T. Proshin, Mikhail Yu. Monakov and Alexander M. Dudchenko

Submitted: 28 March 2018 Reviewed: 15 July 2018 Published: 05 November 2018