RESPIRATORY ACTIVITY IN MEDULLA OBLONGATA and its modulation by

Introduction____________________________________________________________________________

1

From the Neonatal Research Unit

Department of Women and Child Health

Astrid Lindgren Children Hospital

Karolinska Institutet

Stockholm, Sweden

RESPIRATORY ACTIVITY IN

MEDULLA OBLONGATA

and its modulation by

ADENOSINE AND OPIOIDS

Eric HerleniusM.D.

Stockholm 1998

The brainstem and perinatal respiratory activity___________________________________________________________________________

2

This thesis is based on the following papers, which will be referred to in the text by

their Roman numerals:

I Eric Herlenius, Hugo Lagercrantz and Yuji Yamamoto. Adenosine modulates

inspiratory neurons and the respiratory pattern in the brainstem of neonatal rats.

Pediatric Research(1997), 42, 46-53.

II Koichi Takita, Eric Herlenius, Sten Lindahl and Yuji Yamamoto. Actions of

opioids on respiratory activity via activation of brainstemµ-, δ- andκ-receptors;

an in vitro study.Brain Research(1997), 778, 233-241

III Koichi Takita, Eric Herlenius, Sten Lindahl and Yuji Yamamoto. Age and

temperature dependent effects of opioids on medulla oblongata respiratory

activity: An in vitro study in newborn rat.Brain Research(1998), 800, 308-311.

IV Eric Herlenius, Ulrika Åden, Lie-Qi Tang and Hugo Lagercrantz. Adenosine in

the immature rat brain and effects of maternal caffeine intake: development of

respiratory control.Manuscript

V Eric Herlenius and Hugo Lagercrantz. Adenosinergic modulation of respiratory

neurones in thein vitro neonatal rat brainstem.Submitted

Introduction____________________________________________________________________________

3

ABBREVIATIONS

aCSF artificial cerebrospinal fluid

ATP Adenosine-5’-triphospate

Biphasic E Biphasic expiratory neurone (also called Pre-inspiratory)

C4 Cervical ventral root 4

CNS Central nervous system

CPG Central pattern generator

DAGO Try-D-Ala-Gly-[NMephe]-Gly-ol

DPCPX 8-cyclopentyl-1,3,dipropylxanthine

DPDPE [D-pen2,5]-enkephalin

Exp Expiratory related neurone

EPSP excitatory postsynaptic potential

Insp Inspiratory related neurone

Naloxone naloxone hydrochloride

Naloxanazine naloxanazine dihydrochloride

Nor-BNI nor-Binaltorphimine hydrochlorideNTS Nucleus tractus solitarius

PaO2 Partial pressure of oxygen in arterial blood

R-PIA adenosine A1-receptor agonist

(N6-(2-phenylisopropyl) adenosine, R(-) isomer)

Theophylline 1,3,-di-methylxanthine

TTX Tetrodotoxin

U50,488 488 ( trans - (±) - 3,4 - dichloro - N - methyl - N - ( 2 - [ 1 -

pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulfonate)


4

INTRODUCTION .......................................................................................................................................... 5

GENERAL BACKGROUND ...............................................................................................................................5

DEVELOPMENT OF RESPIRATORY CONTROL.................................................................................... 8

Respiratory depression during hypoxia.................................................................................................... 9

OPIOIDS AND RESPIRATORY DEPRESSION....................................................................................................... 11

ADENOSINE AND RESPIRATORY DEPRESSION................................................................................................. 12

THE BRAINSTEM SPINAL CORD PREPARATION................................................................................................ 14

THE PRESENT INVESTIGATION ............................................................................................................ 16

MATERIALS AND METHODS.................................................................................................................. 17

ANIMALS .................................................................................................................................................... 17

BRAINSTEM-SPINAL CORD PREPARATION...................................................................................................... 18

BLIND WHOLE CELL PATCH CLAMP ............................................................................................................... 19

HISTOLOGY ................................................................................................................................................ 21

BAROMETRIC PLETHYSMOGRAPH................................................................................................................. 21

DRUGS....................................................................................................................................................... 22

DATA ANALYSIS AND STATISTICS ................................................................................................................. 23

RESULTS AND DISCUSSION .................................................................................................................... 24

ASPECTS ON METHODOLOGHY..................................................................................................................... 24

Time dependency of opioid and adenosinergic drugs............................................................................. 25

Perinatal age and experiments .............................................................................................................. 25

OPIOIDS AND RESPIRATORY DEPRESSION....................................................................................................... 26

Opioids inhibit respiration byµ- includingµ-1 andκ-opioid receptors ................................................. 26

Temperature and respiratory control..................................................................................................... 27

Do opioids and adenosine interact in respiratory depression?............................................................... 30

ADENOSINE AND RESPIRATORY DEPRESSION................................................................................................. 30

Adenosine modulates respiratory activity by acting directly on brainstem respiration-related neuronal

networks................................................................................................................................................ 31

Adenosine modulates the synaptic activity in brainstem respiratory neurones....................................... 32

Adenosine and postsynaptic depression of respiration-related neurones................................................ 36

Inhibition of expiratory neurones does not abolish the respiratory rhythm............................................ 37

DEVELOPMENT OF RESPIRATORY CONTROL................................................................................................... 39

The neuronal network generating respiratory rhythm does not undergo major changes in the early

perinatal period. ................................................................................................................................... 39

The development of respiratory activity and chronic maternal intake of caffeine................................... 40

GENERAL DISCUSSION............................................................................................................................ 41

CONCLUSIONS........................................................................................................................................... 43

REFERENCES ............................................................................................................................................. 46

ABSTRACT……………………………………………………………………………………………………..56

Introduction____________________________________________________________________________

5

INTRODUCTION

Breathing is a fundamental physiological process produced and controlled by the

nervous system which must be properly set into action from the moment of birth.

Currently it is not exactly known how breathing is initiated and basic mechanisms,

involved in the complex and fine-tuned regulation of respiration are not clearly

delineated yet. A better understanding of normal development of respiratory control

and its disturbances may lead to improved treatment and outcome of clinical

conditions such as repeated episodes of apnoea in preterm babies, sudden infant death

syndrome and postoperative hypoventilation. Hence, the study of mechanisms for

central pattern generation of breathing is of great concern, not least in the perinatal

period, which is the main theme in the present thesis focused on respiratory control

and its modulation by adenosine and opioids.

GENERAL BACKGROUND

It was not until the French revolution that it was understood, during the use of

the guillotine, that mammals do not need their heads to breathe. The French physician

Le Gallois (1770-1814) established that the respiratory centre is localised in the

medulla oblongata in a series of animal experiments (Le Gallois, 1812). This was the

first time that an area within a major subdivision of the brain had been defined

accurately by experimentation as having a specific function (Morton and Garrison,

1991). Le Gallois declared that life in an animal or in any of its organs depends on two

obligatory conditions. One is the integrity of the medulla oblongata and its nervous

output. The second is the circulation of arterial blood to the organ and the medulla

oblongata.

Since the discoveries of Le Gallois our understanding of the body, the brain and

the control of autonomic functions has increased enormously, for reviews see (von

Euler, 1991; Specket al., 1993; Bianchiet al., 1995).

In order to investigate different levels of neuronal networks or single neurones,

animal models have to be used. The anaesthetised or decerebrated cat was the model

of choice for the study of respiratory control from 1920 up to about 1985. Thesein

vivo studies localised the neuronal populations that contain the basic circuitry for

respiratory rhythm in the brainstem as well as the firing pattern, interconnection and

projections of respiratory neurones found in this region (Feldman, 1987; Bianchiet al.,


6

1995). Based on such studies it was determined that the generation of breathing

rhythmogenesis does not critically depend on extrinsic feedback loops or reflexes,

provided the excitability of these mechanisms is kept sufficiently high by adequate

biasing inputs (von Euler, 1980).

During the last decade, thanks to the availability of novel techniques, especially

in vitro en bloc and slice preparations, our understanding has increased concerning

cellular and synaptic physiology of brainstem respiratory neurones. Thesein vitro

preparations make it feasible to examine cellular and sub-cellular levels of respiratory

control. Nevertheless, we still do not know how the neuronal networks in the medulla

oblongata generate respiratory rhythm even though several hypotheses have been

proposed. (Richteret al., 1992; Bianchiet al., 1995; Onimaruet al., 1997; Rekling and

Feldman, 1998). The classical concept of a reciprocal inhibition between two

symmetrical populations of inspiratory and expiratory premotor neurones, the “half-

centre model”, has been abandoned. Instead, the prevailing theories divide the

respiratory cycle into three phases (inspiratory, passive and active expiratory (Richter,

1982). Today, two main hypotheses exist: 1) Site hypothesis: the preBötzinger

complex is the site for respiratory rhythm generation (Smithet al., 1991) and 2)

rhythmogenesis hypothesis: pacemaker or group pacemaker neurones are the cellular

kernel for respiratory rhythm (Reklinget al., 1996) (Onimaruet al., 1988) The

preBötzinger complex in the rostral ventrolateral medulla oblongata seem to have an

obligatory role in respiratory rhythmogenesis, whereas more caudal and dorsal

medullary structures do not. Reciprocal synaptic inhibition between groups of

respiratory neurones is not the cellular basis for respiratory rhythm. This has led to that

neurones intrinsically capable of generating cyclic discharges, i.e., pacemaker

neurones have been proposed as candidates for rhythm generation (Onimaru, 1987) for

reviews see (Rekling and Feldman, 1998) and (Bianchiet al., 1995).

Thus, it has been established that a highly sophisticated central neuronal network

is responsible for the central control of breathing. This central network is able to

continuously adjust breathing to the requirements of the internal and external

environment. Central control implies that the central nervous system is intrinsically

capable of providing the proper timing of muscle activation. Fine-tuning of the central

output is attained by a wide range of vagal afferents from the airways and lungs

relaying sensory information to the nucleus tractus solitarius (NTS) in the dorsal

medulla oblongata. In addition, the arterial baro- and chemoreceptors, both those

relaying via the vagus from the aortic arch and those relaying via the IXth cranial

Introduction____________________________________________________________________________

7

nerve from the carotid bifurcation, send their fibres to the NTS. In addition,

descending inputs from cortex, diencephalon and the cerebellum also influence the

medullary network (Euler and Lagercrantz, 1987) Although suprapontine and sensory

input modulates respiratory rhythm and adapts breathing, these influences only

modulates the ongoing central respiratory rhythm generation. It is important, therefore,

to understand the processes underlying the central respiratory rhythm generation and

its intrinsic control. This thesis focuses on the intrinsic respiratory control system in

the brainstem.

(Modified afterPaxinos and Watson, 1996 and refs.(Arataet al., 1990; Brockhauset

al., 1993).


8

DEVELOPMENT OF RESPIRATORY CONTROL

The ability of central networks to produce rhythmic respiratory motor behaviour

is a well conserved property of the brainstem reticular formation between species

(Borday et al., 1997). Since 1991 an area in the ventral group of respiration related

neurones, the pre-Bötzinger complex, has been considered by most researchers to be

the crucial site of this rhythmogenesis (Smithet al., 1991). This area was identified by

a series of transection studies in thein vitro brainstem spinal cord preparation from

neonatal rats that is the main tool in this thesis. During recent years this area has been

reported to also be importantin vivo and in other species (Paton 1997, (Pierreficheet

al., 1998; Ramirezet al., 1998c). The ventral group of respiratory neurones (VRG)

extends through the whole length of the ventrolateral medulla, partly along the nucleus

ambiguous (Ezure, 1990) but it is mainly in the rostral VRG where bulbospinal

neurones recide (Fig 1). The nucleus tractus solitarius in the dorsal group of

respiratory neurones (DRG) receive afferent input from the lungs and also receive

input from peripheral baro and chemoreceptor. It is thus an important relay for

integrating afferent input that can influence respiration. However, in the rat the DRG

makes little or no contribution to the respiratory drive transmitted from medulla to

spinal motoneurones (Bianchi 1995).

Respiratory rhythm generation is established long before birth, and in the human

foetus respiratory movements already occur at the 11th week of gestation (de Vrieset

al., 1982). These movements are necessary for normal lung development (Kitterman,

1996). Foetal breathing movements are episodic and are progressively more inhibited

towards the end of pregnancy when periods without respiratory movements (apnoea)

dominate (Maloney, 1975 However, from the dramatic moment of birth, these

respiratory neuronal networks need to function and continuously generate a respiratory

rhythm to sustain oxygenation and metabolism. The mechanisms of this transition from

episodic to continuous breathing is still unclear. Respiration must also have the

capacity to respond to changes in the external and internal environments to maintain

body homeostasis. The rhythmic respiratory movements in foetuses, as well as the

breathing in neonates and adults, are governed from neuronal networks in the

brainstem which generate the respiratory rhythm. Thus, during the last decades

researchers have tried to understand how thisnoed vital develops and functions

Introduction____________________________________________________________________________

9

through different experimental approaches, for review see (Mitchell, 1997) and (Speck

et al., 1993).

Respiratory depression during hypoxia

Breathing movements before birth are inhibited by hypoxia (Boddyet al., 1974;

Johnston, 1991). This is functional in foetal life when processes consuming oxygen

have to be turned off if they are not vital for survival (Johnston, 1991; Hanson, 1996).

However, this apnoeic response to hypoxia may be less appropriate after birth. During

the early postnatal period respiratory control is still different than in adults. Both adult

and new-born animals respond to hypoxia with a biphasic change of respiration

(Schwieler, 1968; Lawson and Long, 1983; Runoldet al.Fredholm, 1989). There is an

initial increase of respiration due to stimulation of the peripheral chemoreceptors

(Schwieler, 1968), activation of nervous structures localised rostral to the brainstem

(Eldridge et al., 1981) and structures within the brainstem (Ramirezet al. 1998b).

However, in newborn animals the response to hypoxia is similar to that of the foetus

and the secondary depression of respiration is pronounced and ventilation falls below

normoxic levels within minutes after initiation of hypoxic exposure. During severe

hypoxia or anoxia there are progressive changes in the ventilatory pattern, from

hyperpnoea to a sustained pause of respiration, which is then, after a period of

expiratory apnoea, followed by gasping. Respiratory gasping is characterised by a

series of brief bursts of phrenic activity with sudden onsets followed by a rapid decline

and absence of expiratory activity (Guntheroth and Kawabori, 1975).

What are the brain structure(s) and neuromodulators that are involved in the

hypoxia-induced respiratory depression? Several possibilities have been considered as

mechanisms behind the secondary depression of respiration, for review see (Lawson

and Long, 1983; Neubaueret al., 1990; England, 1993). These hypotheses can be

divided into those stressing inhibitory supramedullary input to the brainstem as vital

for the depressioni.e. (Johnston, 1991; Waiteset al., 1996) (Hanson, 1996; Okadaet

al., 1998) and those emphasising local neurochemical processes in the brainstem

(Moss et al., 1986). Recent findings indicate that even in the reducedin vitro

preparation the hypoxic response is biphasic both at the motor output level and within

the respiratory network (Brockhauset al., 1993; Ramirezet al., 1998a; Ramirezet al.,

1998b). Both afferent and supramedullary input as well as intrinsic brainstem

processes are thus probably involved in this complex behaviour. Diverse structures at


10

several CNS levels are thus involved in the response to hypoxia but the basic

mechanism(s) behind the central inhibition of breathing are still unclear. A proposed

intrinsic brainstem mechanism is a depression of central respiratory neurones mediated

by the release of several neurotransmitters/modulators. These include GABA

(Neubaueret al., 1990) and prostaglandinsPGE2 (REF). Naloxone and theophylline

partially abolish the respiratory inhibition caused by asphyxia, suggesting that opioids

and adenosine are involved (Grunsteinet al., 1981; Chernick and Craig, 1982; Hedner

et al., 1984; Runoldet al., 1989)

Figure 2 Depicts the biphasic ventilatory response to hypoxia (10-15 % O2 in N2).

The inhibitory phase is more pronounced in immature animals. Naloxone or

theophylline can attenuate the inhibition, suggesting that opioids and adenosine are

involved in the secondary inhibition. Administration of dipyridamole, which augments

the endogenous levels of adenosine induces a more pronounced hypoxia-induced

inhibition. (Modified after Lagercrantz, 1987, Grunstein, 1981, Darnall 1985).

0

0,5

1

1,5

-2 3 8

Control

Naloxone /Theophylline

Dipyridamole

Rel

ativ

eve

ntil

ation

Hypoxia min

Introduction____________________________________________________________________________

11

OPIOIDS AND RESPIRATORY DEPRESSION

Opioid-induced respiratory depression is well known from clinical situations as

well as from experimental studies (Yeadon and Kitchen, 1989). Administration of

exogenous opioids is associated with depression of central respiratory activity in

mammals.

Naloxone reverses neonatal depression caused by foetal asphyxia (Chernick and

Craig, 1982). Thus, opioids are thought to be involved in hypoxia-induced respiratory

depression (Mosset al., 1987) Nevertheless, the role of endogenous opioids in basal

respiratory control has not yet been completely established. Some studies using

naloxone have demonstrated that endogenous opioids do not have any significant

influence on the basal regulation of breathing in rats (Steinbrooket al., 1984; Olson,

1987). Others have come to the opposite conclusion (Isom and Elshowihy, 1982).

Recent findings by Greeret al. (Greeret al., 1995) indicate that naloxone has no effect

on medullary respiratory control duringin vivo or in vitro experimental conditions in

newborn rats.

Most studies thus indicate that endogenous opioids are not involved during

eupneic breathing but have an important role during hypoxia and postoperative

hypoventilation. Opioids have also been proposed to be involved in the pathogenesis

of sudden infant death syndrome (Orlowski, 1986; Morinet al., 1992).

Three major classes of opioid receptorsµ, δ, and κ have currently, been

identified, characterised and cloned, all with putative receptor subtypes. All are seven-

transmembrane proteins and members of the G-protein coupled receptor superfamily.

Endogenous opioid peptides with distinctive selectivity profiles exist namely the

enkephalin (µ), endorphin (δ) and dynorphin (κ) groups.µ-receptor binding sites are

present during mid-foetal time and have a high density in cardiorespiratory-related

brainstem nuclei, whereas theδ-opioid receptors primarily appear during the postnatal

period in rats (Xia and Haddad, 1991).

Opioid-induced respiratory depression has been suggested to be caused by direct

actions on the brainstem (Flórez and Hurlé, 1993). Most previousin vivo studies have

suggested thatµ- and δ-opioid receptors participate in opioid-induced respiratory

depression, whileκ-opioid receptors are not involved (Shooket al., 1990; Flórez and

Hurlé, 1993). However,κ- and µ-opioid receptors have a similar distribution in the

brainstem (Mansouret al., 1988) and due to the somewhat unselective receptor

agonists used, it is still debated if onlyµ-receptors are involved in the respiratory

depression caused by opioids. Based on a subdivision intoµ-1 andµ-2 isoreceptors


12

(Pasternaket al., 1980), some authors have claimed that opioid-induced respiratory

depression is mediated byµ-2 receptors (Linget al., 1985; Ling et al., 1986).

However, the exact actions of opioids on respiratory activity via activation ofµ-, δ-

andκ-opioid receptors in the medulla oblongata remain to be elucidated.

ADENOSINE AND RESPIRATORY DEPRESSION

As early as 1929, Drury and Szent-Györygi reported that adenosine can inhibit

respiratory and intestinal movements as well as decrease heart (Drury and Szent-

Gyrörgyi, 1929)). Adenosine is a constituent of all body fluids, including the

extracellular space of the central nervous system. It has multiple effects on organs

and cells of the body (Berne, 1986) Thus, its levels are tightly regulated by a series

of enzymatic steps (Fredholm, 1995). Adenosine can be regarded more as a

neuromodulator in that it does not seem to be stored in vesicles with a regulated

release from nerve terminals. Adenosine is produced by dephosphorylation of

adenosine monophosphate (AMP) by 5`nucleotidase, an enzyme occurring in both

membrane-bound and cytosolic forms (Brundege and Dunwiddie, 1997). Degradation

of intra- and extracellular ATP is the main source of extracellular adenosine

(Dunwiddie and Fredholm, 1997). Specific bi-directional transporters maintain intra-

and extracellular concentrations of adenosine at similar levels. During basal

conditions adenosine levels are 30-300 nM and can rise following stimuli that cause

an imbalance between ATP synthesis and ATP breakdown. Thus, the levels during

ischemia or hypoxia can rise 100-fold (Winnet al., 1981; Fredholm, 1995).

[ATP] i

[AMP] ⇐⇐⇐⇐ [ATP] e.

Inosine⇐⇐⇐⇐ [Adenosine]i ⇔⇔⇔⇔ [Adenosine]e .

bidirectionaltransporter

Figure 3. Schematic representation of the degradation of ATP to adenosine

Introduction____________________________________________________________________________

13

Hypoxia can trigger apnoea in the human neonate (Rigattoet al., 1972; Miller

and Martin, 1992), whereas the adenosine antagonist theophylline blocks hypoxia-

induced depression of breathing in rats (Neylon and Marshall, 1991), rabbits (Runold

et al., 1989) and piglets (Mosset al., 1987; Lopeset al., 1994). The therapeutic effect

of theophylline on neonatal apnoea has thus been suggested to be due to its

antagonistic action on adenosine receptors (Lagercrantz Yet al., 1984; Darnall, 1985;

Hedneret al., 1985). Several lines of evidence indicate that adenosine can inhibit

respiration-related neurones in the brainstem (Eldridgeet al., 1983; Thomaset al. ,

1994). However, adenosine also decreases metabolism and oxygen consumption

through inhibiting lipolysis and non-shivering thermogenesis (Ballet al., 1996).

Neonatal animals respond to hypoxia with decreased body temperature. Thus, it has

been suggested that the main effect of adenosine in depressing breathing is indirect by

a decrease of oxygen consumption (Lagercrantzet al., 1986).

To date four distinct receptor subtypes have been identified by cloning, denoted

A1, A2a, A2b and A3-adenosine receptors. All subtypes belong to the family of rhodopsin

G-protein-coupled receptors (Fredholm, 1995). Basal levels of adenosine can act on

A1- and A2a–receptors whereas A2b-receptors only are activated at pathological

adenosine levels. A2a receptors are coupled to GTP-binding (G) proteins classified as

Gs, because of their stimulatory effect on adenylyl cyclas, and mainly expressed in

dopamine-rich regions such as the striatum. Adenosine A1-receptors are coupled to Gi(1-

3) (inhibitory to adenylyl cyclas) or Go (no effect on adenylyl cyclas) proteins and are

ubiquitously expressed in the CNS with higher expression in regions such as the

cortex, hippocampus and cerebellum (Fredholm, 1995). A1-receptors have been

characterised by behavioural, biochemical and anatomical studies, for a review see

(Dunwiddie and Fredholm, 1997). A1-receptors can modulate synaptic transmission in

the CNS through several different mechanisms. Among these are decreased

neurotransmitter release (Mynlieff and Beam, 1994) and reduction of neuronal

excitability through hyperpolarisation of neurones (Thompsonet al., 1992). Adenosine

have been demonstrated to modulate cardiovascular control through its action in the

rostral ventrolateral medulla (Thomas and Soyer, 1996). Responses to adenosine are

different even in neuronal populations using the same transmitter, which makes it

difficult to directly extrapolate data obtained from one group of neurones to another.

Thus, a modulation shown for parts of a neuronal network may not be applicable when

all its components are examined.

Adenosine is also an important neuromodulator in foetal life (Bissonetteet al.,

1990; Koos and Matsuda, 1990) and at birth (Irestedtet al., 1989). Changes of


14

adenosinergic tonic activity due to caffeine, an adenosine antagonist, may interfere in

the communication between developing neurones leading to long term sequele.

Caffeine, is the most widely consumed neuroactive substance worldwide. Negative

effects on fertility, birthweight, risk for prematurity and congenital malformations of

high doses of caffeine have been demonstrated in animals, for review see (Nehlig and

Debry, 1994). There is however little evidence that normal human caffeine

consumption has any of these consequences.

Does maternal intake of adenosine antagonists, such as caffeine affect the

development of respiratory rhythm generation and control during foetal and neonatal

life? High doses of caffeine intake during gestation or the early postnatal period affect

postnatal behaviour in rats (Holloway and Thor, 1982; Guillet, 1990; Devoeet al.,

1993; Guillet and Dunham, 1995). Effects of more moderate prenatal caffeine intake

on postnatal behaviour have also been studied, and several investigators have

described behavioural hyperactivity in developing rodents (Sobotka, 1979; Sinton,

1981; Holloway Jr, 1982; Holloway Jr and Thor, 1982). If low doses of caffeine

during gestation can induce long-term changes in adenosine receptor expression and if

the development of respiratory control is affected this may have important clinical

implications.

THE BRAINSTEM SPINAL CORD PREPARATION

Studies on respiratory control and the highly sophisticated neuronal networks

that generate this complex behaviour can be performed at different levels: whole-body,

organ, neuronal networks, cellular, synaptic and molecular. During the last 15 yearsin

vitro experimental preparations have proved invaluable in unravelling the mechanisms

of central respiratory control. Among the powerful advantages ofin vitro preparations

are: 1) direct access to neurones of interest; 2) mechanical stability, allowing

intracellular recordings with whole-cell patch techniques; and 3) improved control of

the environment surrounding the neurones.

A brainstem-spinal cord preparation from neonatal rats was first used by Suzue

and co-workers (Suzue, 1984). This en bloc brainstem spinal cord preparation

generates respiration-related rhythmic activity for several hours when perfused with

oxygenated artificial cerebrospinal fluid (aCSF). Compared to thein vivo situation, the

respiratory frequency is low. Inspiratory-phase dischargein vitro is rapidly peaking-

slowly decrementing instead of a short duration, augmenting discharge envelope

(Smithet al., 1990). The transformation of the respiratory motor patternin vivo to that

Introduction____________________________________________________________________________

15

of the isolated and reduced brainstem spinal cord preparation can be explained by

several facts: 1) it does not have suprapontine inputs and can be further reduced by

removing the pons; 2) removal of vagal mechanosensory afferent input; 3) low

temperature (25-32oC) (Smithet al., 1990; Onimaru, 1995a).

Respiratory rhythm generation in neonatalin vitro models differs from that of

adult in vivo models. In this neonatal model Chloride-mediated inhibition is not

necessary for the respiratory rhythm whereas it probably is in the adult (OnimaruArata

and Homma, 1990; Feldman Smith and Let al.iu, 1991; Onimaruet al., 1997;

Ramirezet al., 1997). However, the isolated respiratory network in the brainstem is

sufficient to generate the essential features of respiratory activity at the cellular and

system levels that have previously been described for the intact respiratory system.

The relevance of neuronal mechanisms studied in this reduced preparation to

those operating in the intact neonatal or adult rat has been determined by several

studies during the last decade, for reviews see (Smithet al., 1990; Onimaruet al.,

1997).

Synaptic events during the respiratory cycle have not been thoroughly analysed

in any neonatal mammalian species, so the correspondence between the synaptic

events producing the cycle phasing in the neonatal and adult system remains to be

established (Smithet al., 1990) The studies that have been performed reveal

remarkable similarities in the synaptic interactions between respiratory neurones

although recordings fromin vivo cat andin vitro rodents were compared. (Ramirezet

al., 1997).

Neonatal rats, as all mammals, are born with a functional central pattern

generator for respiration. However, the CNS and its connections with the internal and

external environments develops before and after birth. As described in the previous

pages it is known that both adenosine and opioids have different effects on respiration

in the neonate compared to the adult mammal. Are these changes in modulatory

effects initiated at the moment of birth, during the first postnatal days or are they

dependent on progressive maturation of neuronal networks, receptor expression and

affinity? We have in the following studies used the brainstem spinal cord preparation,

complemented within vivo andin situ techniques, to address some of these questions.

The PresentInvestigation

_____________________________________________________________________

16

THE PRESENT INVESTIGATION

The overall aim of the present study was to further elucidate the perinatal development

of central respiratory activity and its control. Specifically, the aims where to:

• investigate if there is a transition at birth of the properties of the isolated neuronal

network that generate respiratory rhythm. (IV)

• determine if adenosine and opioids inhibit respiration by acting directly on the

central neuronal networks generating respiratory activity. (I-V)

• determine if the adenosinergic and opioidergic modulation of respiratory activity in

the medulla oblongata changes during the first days after birth. (I, III-V)

• investigate if chronic maternal intake of caffeine (a typical adenosine antagonist),

during gestation and early postnatal life, alters the development of respiratory

control. (IV)

• investigate the mechanisms behind and at which level adenosinergic modulation of

central respiratory activity occurs, i.e. pre- vs. postsynaptically.(I and V)

Material and Methods____________________________________________________________________________

17

MATERIALS AND METHODS

ANIMALSRats were bred by certified animal supplier and stored at the animal facilities

according to local regulations. Neonatal rats were kept with their mothers until the

experiments started. Pups were examinedin vitro at postnatal age 0-4 days and at

embryonic (E) day 18-21. Rat brains were examined at E14, E18, E 21, exactly 2

hours and 24 hours after vaginal delivery and postnatal day (P)3 and P7.

In the experiments performed in paper IV, examining the development of

respiratory control, timed pregnant rats were used. The day when a vaginal plug was

found was designed as embryonic day 0. The regional animal ethics committee

approved the experiments, which followed the European Community regulations.

Paper Questions n Methods

I Early postnatal adenosinergic

modulation of respiratory activity62 Brainstem spinal cord

preparation (BSP)

II Opioid-receptors 156 BSP

III Opiods-temperature and age 273 BSP

IV Development of respiratory control

and its adenosinergic modulation

Development of adenosine A1-

receptors and maternal caffeine

80

40

200

BSP

Barometric plethysmograph

In situ hybridisation and

receptor autoradiography

V Mechanisms of central adenosinergic

respiratory modulation?111 BSP

Blind whole cell patch clamp

An extended description of the blind whole-cell patch clamp technique, the

accompanying histological method as well as thein vivo experiments follows below.

The other methods used are described in the papers included.

The brainstem and perinatal respiratory activity____________________________________________________________________________

18

BRAINSTEM-SPINAL CORD PREPARATION

The in vitro experiments were performed on the brainstems and spinal cords of

newborn (0- to 4-day-old) Bkl:S-D or Wistar rats. Under deep ether anaesthesia, the

brainstem-spinal cord was dissected and isolated as described previously (Suzue,

1984). The brainstem was then rostrally decerebrated between the VIth cranial nerve

roots and the lower border of the trapezoid body. This preparation was subsequently

transferred to a 2-ml chamber, where it was continuously perfused at a rate of 3.0-3.5

ml min-1 with artificial cerebrospinal fluid (aCSF) equilibrated with 95% O2 and 5%

CO2at 26-28oC to give a pH of 7.4. Foetal rats were obtained from plugged dams by

caesarean section. Respiratory activity was recorded employing suction electrodes

applied to the proximal ends of cut C4 or C5 ventral roots containing respiratory

motoneurone axons that innervate the intercostal muscles and the ribcagein vivo. (Fig.

2). The phasic C4/C5 activity corresponds to phrenic nerve discharges (Suzue, 1984;

Smith et al., 1990). The regularity of respiratory activity was quantified as coefficient

of variation (CV) of the interval between C4 burst discharges.

For further details see paper I-V

Figure 4 Brainstem-spinal cord preparation


19

BLIND WHOLE CELL PATCH CLAMP

Whole cell recordings from respiratory neurones were obtained using the

modified blind patch clamp technique (Blantonet al., 1989; Onimaru and Homma,

1992). Briefly, patch pipettes were pulled in one step from borosilicate glass

(GC100TF-10, outer diameter 1.0 mm, with a filament; Clark Electromed, Reading,

UK) using a vertical puller (PE-2; Narishige, Tokyo, Japan). The electrode tips had

inner diameters of 1.2-2.0µm and a DC resistance of 4-8 MΩ. The electrodes were

filled with a solution consisting of (mM): potassium gluconate 120,

ethylenebis(oxonitrilo)-tetraacetate (EGTA) 10, 4-(2-hydroxyethyl)-1-

piperazineethane-sulphonic acid (HEPES) 10, CaCl2 1, MgCl2 1 and Na2-ATP 1. KOH

was used to adjust the pH to 7.3. In a number of experiments this electrode solution

also contained 0.2 % Lucifer Yellow (LY; Sigma, St. Louis, Mo, USA). The solution

was routinely filtered through a Millipore filter (pore size 0.45µm) immediately

before its introduction into the electrodes.

A small area of thepia mater was removed using a glass needle and the

electrodes were inserted through this area into the ventral medulla. A slight positive

pressure (5-15 cm H2O) was applied during advancement of the electrode to keep the

electrode tip clean. Extracellular signals and intracellular membrane potentials were

measured with a voltage clamp amplifier (Nihon Koden, CEZ-311). Respiration-

related neurones were sought in RVL (Arataet al., 1990) in the region of the ventral

respiratory group (Richteret al., 1992), while monitoring extracellular signals

amplified with a loudspeaker (Onimaru, 1995b). In whole cell experiments, when such

a respiration-related neurone was found, a slight negative pressure was applied. The

resulting formation of a gigaohm seal (>1GΩ) was monitored and confirmed by

applying a hyperpolarising current pulse (0.1 nA; duration, 30 ms 2 Hz). When this

seal had been established, the pressure was returned to zero. Rupture of the cell

membrane was achieved by applying negative pressure (50-80 cm H2O) often together

with a single hyperpolarising current pulse (0.5-0.8 nA; duration 30 ms). When whole

cell recording had been established the pressure was rapidly returned to zero ±2-3 cm

H2O. Series resistance (20 - 50 MΩ) was compensated by a bridge balance circuit. The

voltage deflections induced by 1 Hz 30 ms hyperpolarising pulses were monitored and

compensated for by eye.


20

Neurones were identified and classified on the basis of their characteristic firing

patterns and the temporal correlation of this activity to the respiratory cycle of C4/C5

activity. Inspiratory neurones (Insp) receive excitatory synaptic input and discharge

action potentials during inspiratory phrenic (C4/C5) activity. Expiratory neurones

(Exp) discharge action potentials between the inspiratory phases and are inhibited

during the inspiratory phase (Shao and Feldman, 1997). Biphasic expiratory neurones

(also classified as Preinspiratory by Onimaruet al.) are characterised by pre- and

postinspiratory excitation and inspiratory-related inhibition (Onimaruet al., 1990;

Smithet al., 1990).

Inspiratory neurones were further classified into three subtypes, according to

previous classifications performed with this preparation (Onimaruet al., 1996;

Onimaru et al., 1997). Type I neurones (Insp I) receive excitatory postsynaptic

potentials (EPSPs) prior to the onset, as well as after the termination of C4 activity,

whereas type III neurones (Insp III) are hyperpolarised by synchronised inhibitory

postsynaptic potentials (IPSPs) during the pre- and postinspiratory phases. Insp

neurones, which only exhibited EPSPs during the inspiratory phase and no

hyperpolarisation during the pre- or postinspiratory phase, were classified as type II

neurones (Insp II ).

Respiratory cycle

Neuronal type Inspiratory Peri-inspiratory Expiratory-I Expiratory-II

Insp type-I ++ (+) 0 0Insp type-II ++ 0 0 (+)Insp type-III ++ - 0 0Expiratory-t - + + +Expiratory-l - - 0 +Biphasic E - + 0 0

Table 1 Respiration-related neurons in the brainstem spinal cord preparation, here

categorised in relation to the C4 inspiratory activity according to Onimaruet al.

(Onimaru 1997)- = hyperpolarised, Inspiratory (Insp), Biphasic expiratory (Biphasic

E), tonic Expiratory (Expiratory-t), late Expiratory (Expiratory-l)

The current-voltage (I-V) relationship was determined by injection of an inward

current (0.02-0.08 nA; duration 100 ms) during silent phases between bursts or during


21

negative holding potentials (-50 mV) in the case of certain expiratory neurones. Input

resistance (Rm) was calculated from the slope of a least-squares regression line fitted to

the data. In Exp neurones, the frequency (action potentials / min) of expiratory

neuronal activity was examined. During the experiments, signals were displayed on a

chart recorder, monitored using an oscilloscope, digitalised (Digidata 1200B, Axon

Instr., Foster, CA, U.S.) and stored on DAT tape (RD-120TE, TEAC, Tokyo, Japan)

or a hard disc for off-line analysis.

HISTOLOGYAfter completing thein vitro recordings, those preparations containing neurones

filled with Lucifer Yellow were removed from the recording chamber and placed in a

solution of cold (4OC) Lillie solution (10% formalin in phosphate buffer, pH 7.0) for

at least 48 hours. The preparation was then rinsed and placed serially for 10 min each

in 5, 10 and 15% sucrose in 0.1 M phosphate buffer (PB, Sorensen-Gomori, pH 7.2).

The sample was subsequently stored in the 15% solution at 4oC for at least 12 hours

before 70-µm sections were cut on a cryostat. Sections were mounted on glass slides,

coated with PBS and propriogallate. The intracellularly marked Lucifer Yellow-

neurones were photographed and reconstructed with the aid of a camera lucida

attached to a fluorescence microscope (Olympus, Tokyo, Japan). Counterstaining was

performed with 2% neutral red. After dehydration in a series of solutions containing

increasing ethanol concentrations, sections were clarified with xylene and a cover was

placed on them.

BAROMETRIC PLETHYSMOGRAPHIn vivo ventilation was monitored with a barometric plethysmograph, a procedure

which allows non-invasive recording of respiration in unanesthetized animals

(Drorbaugh and Fenn, 1955). This method is based on the fact that warming a gas

maintained at a constant volume increases its pressure. Thus, during inspiration,

pressure inside the chamber increases and during expiration it decreases. The animals

were placed in a plexiglass chamber (160 ml in volume) which, together with an

identical reference chamber, was connected to a highly sensitive differential pressure

transducer (Validyne DP103). Pressurised gas from gas tubes was continuously fed to

the recording chamber and removed with a vacuum pump. The resistance of the inlet

and outlet tubing was adjusted with needle valves in order to maintain approximately

atmospheric pressure in the recording chamber. The rate of gas flow through the

recording chamber was about 1.2 l min-1. The time constant of the recording system


22

was 0.2 seconds. The pressure signal was amplified and recorded by an on-line

computer. The measurements of breathing were only semiquantitative because core

body temperature was not measured and because of the short time constant of the

recording system. Animal and chamber temperatures were recorded using digital

thermometers with an accuracy of 0.1oC. At the onset of the experiment the chamber

temperatur was kept at 29.5 ± 0.5oC via a thermoradiator and a heating pad.

DRUGSThe pH was assessed and adjusted to 7.4 with 95% O2 and 5% CO2 prior to bath

application of drugs. Sufficient time for achievement of the steady-state drug response,

determined for each drug used was allowed to elapse before determination of the drug

response. In paper IV dams were given caffeine (0 or 0.3g/L) in the drinking water from

embryonic day 2 (E2) throughout gestation and postnatal life. The daily intake of water

was measured in all litters. Blood from five litters were collected in heparinized plastic

tubes and centrifuged. Plasma concentrations of caffeine and its metabolites theophylline,

theobromine and paraxanthine were analysed using HPLC. In paper II 17 animals received

a subcutaneous injection (s.c.) of naloxanazine 35 mg/kg ( =5 ml naloxanazine solution/kg)

and 17 animals received saline 5 ml/kg s.c. 22-26 hours after these injectionsin vitro

studies were performed. See Table 3 for drugs used.


23

Table 3. Major pharmacological tools used in this thesis

Compound Selectivity

Adenosinergic

R-PIA A1-adenosine receptor agonist

Dipyridamol nucleoside transport inhibitor

DPCPX A1-adenosine receptor antagonist

Theophylline Adenosine receptor antagonist

Caffeine Adenosine receptor antagonist

Opioidergic

Morphine opioid receptor agonist

DAGO µ-opioid receptor agonist

DPDPE δ-opioid receptor agonist

U50,488 κ-opioid receptor agonist

Naloxone opioid receptor antagonist

Nor-BNI κ-opioid receptor antagonist

Naloxanazine µ-1 opioid receptor antagonist

DATA ANALYSIS AND STATISTICS

Off-line analysis was performed using a personal computer and the commercially

available programs Axoscope (Axon Inc., Foster, CA, USA), Origin (Microcal

Software Inc., Northampton, MA, USA) and JMP (SAS Inst. Inc., Cary, NC, USA).

The results are presented as means ± standard deviations or standard errors of the

mean when small or non-normally distributed data are presented. After analysis of the

variance by theF-test, statistical analysis was performed using the multivariate

analysis of variance (MANOVA) repeated measure design, two-tailed paired Student’s

t-test or Wilcoxon's signed-rank test (when variances were unequal). The differences

between several independent means were analysed by comparing all pairs using the

Tukey-Kramer HSD test. Spearman rank nonparametric correlation was performed on

measured variables with respect to postnatal age, in order to evaluate a possible

dependency of the results on age. A P value of <0.05 was considered to be statistically

significant.

Brainstem perinatal respiratory activity___________________________________________________________________________

24

RESULTS AND DISCUSSION

ASPECTS ON METHODOLOGHY

The region where respiration-related neurones in the neonatal rat are located is in

the ventrolateral reticular formation at depths of 50-500µm. In the brainstem spinal

cord preparation there are substantial gradients of PO2, PCO2, K+ and pH from the

surface (Brockhauset al., 1993; Okadaet al., 1993; Voipio and Ballanyi, 1997) to the

respiratory network but the oxygenation permits the neurones to operate under

conditions of aerobic metabolism (Brockhauset al., 1993). Furthermore, the brainstem

spinal cord preparation is sensitive to changes in pH and oxygenation and responds in

a similar manner at the neuronal and motor output levels, as do animalsin vivo

(Ramirezet al., 1998b). The preparation is thus used for studying cellular mechanisms

and the localisation of central chemosensitivity as well as the biphasic respiratory

response to hypoxia (Kawaiet al., 1996; Ramirezet al., 1998b). The preparation is

also suited for detailed pharmacological studies of the respiratory centre in

anaesthesia-free conditions. Drugs can be applied at defined concentrations to the

regions of interest and simultaneous recordings of respiratory output and from

individual neurones made.

Although it has several advantages, thein vitro system is a reduced preparation

with altered parameters of neuronal function including CNS temperature (25-30°C).

Several parameters of neuronal function, including the kinetics of membrane currents

(Hille, 1984) are temperature dependent (Smithet al., 1990). Hypothermia can

attenuate or enhance the action of neuromodulators/drugs affecting respiration (Vitez

et al., 1974; Puiget al., 1987). The temperature therefore has to be considered when

performing experiments using this preparation.The size of thein vitro preparation is a serious limitation since substantial

gradients for oxygen, carbon dioxide and pH may alter the condition of the tissue and

its viability (Mitchell, 1993). Thein vitro preparation is highly reduced by the removal

of afferents and suprapontine inputs. This is desirable in that it excludes several

compensatory mechanisms, i.e., cardiovascular changes, that may complicate data

interpretation. However, this also alters the state of the central pattern generator by

removing tonic inhibitory and excitatory input. Likewise, because of the artificial

Results and Discussion__________________________________________________________________________

25

environment of the neurones, variables such as pH, oxygenation and steady state have

to be taken into consideration when determining effects of drugs.

Thus, data have to be cautiously interpreted and compared with data fromin vivo

models to determine the relationship between the nervous system behaviourin vitro

with that of in vivo systems. We demonstrate below how results depend on time,

temperature as well as perinatal age when performing experiments in this model using

mammals still in a developmental phase.

Time dependency of opioid and adenosinergic drugs

The drug effect has been evaluated after 5 minutes in several of the previous

studies. We demonstrate in the present studies (I-III and V) that steady state effect of

both adenosinergic and opioidergic drugs can take as long as 10-20 minutes to establish.

There are several possible explanations for this:a) Drug kinetics: R-PIA is highly

lipophilic, and thus it is possible that R-PIA binds to fat-rich glia before a gradient and

slow diffusion establishes steady state concentrations within the region of the respiratory

neurones, 50-600µm below the ventral surface;b) Receptor recruitment: recent studies

indicate that some G-protein linked receptors increase at the cell surface when

stimulated. A1-receptors are recruited from the cytoplasm to the surface of neurones after

stimulation (Ciruelaet al., 1997). In addition, it has recently been reported that activation

of dopamine D1-receptor, also G-protein coupled, induces a time- and dose-dependent

recruitment of D1-receptors to the cell surface (Brismaret al., 1998). Thus, an alternative

explanation for the time dependency could be that the full effect of ligands first is

reached when ligand-induced aggregation of receptors to the cell surface is completed.

However, our studies cannot determine the mechanisms for this time dependency, but

nevertheless underline the importance of taking time into account when determining drug

effects in this preparation.

Perinatal age and experiments

The inhibitory effect of morphine on the respiratory rhythmic activity increased

with postnatal age (P0-P4) (III) while the depressant effect of adenosine on respiration

decreased during both foetal (E18-E21) and postnatal life (P0-P4) (I, IV and V). Age

has a significant effect on both control respiratory activity (I and IV) and the effects of


26

drugs (I-V) during the first postnatal days. This fact has been neglected in several

studies using thein vitro brainstem spinal cord preparation. (Murakoshi, 1985; Greer

et al., 1995). These findings will be discussed in the context of development of

respiratory control later on.

OPIOIDS AND RESPIRATORY DEPRESSION

Opioids inhibit respiration by µµµµ- including µµµµ-1 and κκκκ-opioid receptors

To study the effects of opioids on brainstem opioid receptors effects of theµ-

receptor agonist DAGO, theδ-receptor agonist DPDPE and theκ-receptor agonist

U50,488 were evaluated in the brainstem spinal cord preparation (II and III). The

opioid receptor antagonists naloxone (µ) and naloxanazine (µ-1) were also used.

DAGO reduced respiratory frequency and inspiratory time in a concentration-

dependent manner and at high concentrations (10µM) caused a reduction of peak

integrated C4 amplitude. Theµ-1 receptor antagonist naloxanazine shifted the

concentration- respiratory frequency response curve for DAGO to the right (p<0.05).

Thus, higher concentrations of DAGO were needed to give an equivalent respiratory

depression as that in animals not pre-treated with theµ-1 antagonist. DPDPE had no

effect on respiratory activities whereas U50,488, like DAGO, reduced respiratory

frequency and amplitude of integrated C4 in a concentration-dependent manner. Thus,

µ-opioid receptors, including theµ-1 receptors are involved in respiratory frequency

reduction whereasκ-opioid receptors are involved in reduction of both respiratory

frequency and respiratory amplitude.δ-opioid receptors do not seem to participate in

respiratory modulation in the neonatal rat.

The finding that aκ-opioid receptor agonist depresses respiration is in conflict

with the findings of Greeret al. (Greeret al., 1995). However, it should be noted that

these authors only conducted their measurements 5 min after drug application (Greer

et al., 1995) while our evaluations were performed after 20 min. This methodological

difference most certainly explains the discrepancy between the studies.

Our results are in agreement with previous findings in adult rats in which

microinjection ofκ-opioid receptor agonists into ventral medullary structures reduced

VT and respiratory frequency (Hassen, 1984). Nevertheless, most of the previousin

vivo studies (Leightonet al., 1987; Yeadon and Kitchen, 1990; Dosaka-Akitaet al.,


27

1993) have indicated thatκ-opioid receptors do not participate in opioid-induced

respiratory depression. This discrepancy between the results of thesein vivo studies

and the presentin vitro study suggest that suprabulbar structures may modify

medullary κ-receptors-mediated respiratory depressant effects. If=κ-receptors are

involved in the respiratory depression of opioids this is important knowledge in the

development of future opioidergic analgesia without respiratory depressanteffects.

Temperature and respiratory control

The temperature normally used in the different mammalianin vitro preparations

is 25-26°C. The low temperature compared toin vivo physiological conditions is used

to decrease oxygen consumption and to sustain regular respiratory activity for long

periods of time. Reduced temperature may have consequences beyond the desired

effects of reduced metabolic rate and prolonging the viability of the preparation. Low

temperatures may alter membrane fluidity, thereby altering membrane bound proteins

including receptors and ion channels. (Mitchell, 1993). In paper III we examined the

possible influences of temperature on respiratory depression induced by opioids. We

demonstrate that lower temperature markedly reduced the ability of morphine (Fig. 6)

and DAGO to decrease respiratory frequency. Changes in temperature did not affect

the dose-response curve forκ-receptor activation by U50,488. We conclude that the

respiratory effect of morphine and DAGO via activation of medullaryµ-opioid

receptors is temperature-dependent.


28

Figure 5 Temperature dependent respiratory depression by morphineThe influence of change in temperature on the C4 activity (upper trace)

integrated C4 (lower trace) and their response to morphine 50µM. A) the respiratory

activities recorded from thein vitro preparation in standard solution at 25.5°C. B) 20

minutes after increasing temperature to 28.5°C) 30 min after perfusion with morphine

50µM D) 30 min after decreasing chamber temperature to 25.5°C.


29

The reduced respiratory frequency, caused by morphine in our study, is in

agreement with what has been previously reported(Murakoshi, 1985). Hitherto, not

much has been described about effects of temperature on opioid receptor mechanisms.

It has been proposed that the affinity of theµ-receptor for opioids can be modified at

different temperatures (Garauletet al., 1992). Since receptor proteins can undergo

conformational changes due to temperature changes and, hence, alter their affinities

for the opioid molecule, this could explain our findings. However, the affinity of

naloxone for opioid receptors is not temperature dependent (Puiget al., 1987;

Kuemmerle and Makhlouf, 1992). This concords with previous studies of intestinal

muscle preparations, in which the potency of DAGO (µ-agonist), DPDPE (δ-agonist)

and morphine decreased with a lower temperature while in the range from 37-30°C

(Puiget al., 1987; Garauletet al., 1992).

The opioid receptor is a classic seven transmembrane receptor with three

extracellular loops deciding ligand specificity (Befort, 1997).µ- andδ-agonists require

the third extracellular loop for binding.κ-selectivity lies primarily within the 1st and 2nd

extracellular loop which may gain structural stability from the cysteine-cysteine bridge

connecting these extracellular receptor domains (Traynor, 1996; Befort, 1997). A

speculative explanation of the selectiveµ-receptor temperature dependency could be

that the third extracellular loop, which is not stabilised with a disulphide bridge, is

more susceptible to changes in the temperature. Thus, instability of the third

extracellular loop of the opioid receptor may lead to conformational changes with

temperature and alter the affinity forµ- andδ-agonists.

Our observations of temperature dependent opioid receptor function are of

clinical interest since opioids are frequently used in patients, not least during

hypothermia for cardiopulmonary bypass surgery. In the clinical setting analgesia and

not respiratory depression is the goal. With large doses of opioids administered during

hypothermia, significant side effects will appear at normothermia. Linget al. (Ling et

al., 1985) suggested that opioid-induced analgesia and respiratory depression are

mediated via activation of different subtypes ofµ-opioid receptors. According to their

in vivo study, opioid-induced analgesia was linked toµ1 opioid receptor activation and

respiratory depression was linked toµ2. However, using this brainstem spinal cord


30

preparation we demonstrate that the medullaryµ1 opioid receptors also participate in

respiratory frequency reduction (III).

This observation links analgesia with respiratory depression. Clinically it is well

known that the respiratory depressant effect of opioids does not appear until significant

pain relief is achieved. Such a clinical effect is straightforward and logical if the two

modalities, analgesia and respiratory depression, are mediated via the same receptor.

Our results are not necessarily in disagreement with the observation of Linget al., who

reported thatµ2-receptors have an impact on respiratory timing. However, it challenges

the assumption that pain relief viaµ-receptors can, one way or another, be separated

from respiratory depression (Linget al., 1985).

Do opioids and adenosine interact in respiratory depression?

Some of the pharmacological effects of morphine seem to be due to the

presynaptic modulation of transmitter release of substances such as acetylcholine,

substance P and adenosine (Jessell and Inversen, 1977; Sawynoket al., 1989; Taguchi

et al., 1993). In the spinal cord a component of the antinociceptive action of morphine

is due to the release of adenosine (Sollevi, 1997; Sawynok, 1998). Thus to test the

hypothesis that part of the effect of morphine on respiration is mediated by indirect

effect of other transmitters / modulators, we tried to reverse the morphine-induced

respiratory depression by adding adenosine antagonists, substance P and acetylcholine.

Acetylcholine (10µM) and substance P (50 nM) could partly, but not fully, reverse the

morphine-induced reduction of respiratory frequency via respiration-related structures

in the rostral medulla (Takita, Herleniuset al. unpublished observations). However,

the adenosine receptor antagonist theophylline (100µM) does not affect morphine-

induced respiratory depression at all. This would suggest that acetylcholine, substance

P and adenosine are not crucially involved in morphine-induced respiratory

depression.

ADENOSINE AND RESPIRATORY DEPRESSION

Adenosine levels increase during hypoxia and have been proposed to be involved

in hypoxia-induced depression of breathing due to its inhibitory action on the central


31

nervous system. The following results describe where and how adenosine can

modulate respiration through respiration-related neurones in the brainstem.

Adenosine modulates respiratory activity by acting directly on brainstemrespiration-related neuronal networks

In paper I we demonstrate that R-PIA and dipyridamole decrease the activity of

inspiratory neurones (I-neurones) and the C4 respiratory burst rate. Theophylline or

DPCPX reverse the effects of both R-PIA and dipyridamole on C4 respiratory rate and

intra-burst fq of I-neurones. Thus, adenosine depresses both the I-neurones in the RVL

and the respiratory motor output. This depression of I-neurones and respiratory rate

can be abolished by theophylline primarily through a blockade of medullary adenosine

A1 receptors. These results were confirmed and also shown to be valid in the foetal

period in papers IV and V.

The studies presented in this thesis, demonstrate that endogenous adenosine

modulates respiration by acting directly on the neuronal network for breathing

rhythmogenesis (I, IV and V). This does not exclude the possibility that adenosine also

may inhibit respiration via an indirect effect on body temperature and metabolism

(Lagercrantzet al., 1986). However, as indicated in a recent study, the decrease in

metabolism that occurs during hypoxia does not seem to be responsible for the

decrease of respiration induced by hypoxia (Rehanet al., 1996). Our findings confirm

and verify previous suggestions that adenosine modulates respiration through an action

on medullary respiratory networks (Eldridgeet al., 1985; Thomaset al., 1994).

Theophylline and caffeine are adenosine antagonists that cause ventilation to

increase when given systemically (Aranda and Turmen, 1979; Eldridgeet al., 1983).

Furthermore, theophylline can attenuate both the hypoxia-induced depression of

respiration in vivo (Darnall, 1985; Runoldet al., 1989) and the depression of C4

motoneuronal dischargein vitro (Kawai et al., 1995). This indicates that part of the

respiratory decrease induced by hypoxia (Cross and Warner, 1951; Haddad and

Mellins, 1984) can be explained through increased adenosine levels and A1-R

mediated inhibition of respiration-related neurones in the RVL. This finding is

important since two of the most widely used drugs to treat apnoea of prematurity are

theophylline and caffeine (Aranda and Turmen, 1979). This also implies that the

decreased incidence of neonatal apnoeas during theophylline treatment is mainly due

to the antagonistic effect of theophylline on A1-receptors in the medulla oblongata.


32

Adenosine modulates the synaptic activity in brainstem respiratory neurones

What are the mechanism(s) behind adenosinergic depression of respiration? After

having found that adenosine acts on respiratory neurones in the rostral ventrolateral

medulla to depress respiratory output we wanted to clarify the mechanisms behind this

depression. To be able to determine synaptic events and changes in membrane

properties of individual neurones intracellular recordings were necessary. To achieve

this goal, the modified blind whole cell patch-clamp technique was used on respiratory

neurones identified by their characteristic firing patterns and correlated in time to the

respiratory cycle of C4/C5-activity. This allowed us to investigate adenosinergic

effects on membrane potential and resistance of RVL neurones, simultaneously

recording changes in C4 respiratory output. Lucifer Yellow labelled neurones were

located 50-500µm below the closest ventral surface and ventral of the ambiguous

nucleus, in agreement with previous findings (Arataet al., 1990). Camera Lucida

reconstruction of the major respiratory neurone subtypes found in vitro are illustrated

in Fig. 6.

Figure 6. Camera Lucida reconstruction of respiratory neurones: biphasic expiratory (Pre-

I) and expiratory neurone in proximity of an inspiratory neurone.

In paper V, we demonstrate that R-PIA consistently causes a reduction of

spontaneous synaptic activities during the expiratory phase in all RVL neurones

examined. Figure 7 (modified from paper V) depicts the characteristics of major


33

groups of respiration-related neurones found. The A1-R agonist and antagonist induced

modulation of excitatory postsynaptic potentials did not correlate with changes in

membrane potential (Em) or input resistance (Rm). This indicates that adenosine

depresses respiration through presynaptic adenosine A1-receptor mediated inhibition of

synaptic transmission in the medulla oblongata. This is in agreement with the now

well established role of adenosine as a regulator of CNS synaptic transmission

(Snyder, 1985; Brundege and Dunwiddie, 1997; Dunwiddie and Fredholm, 1997).

Several possible mechanisms exist for by which presynaptic A1-receptor can decrease

transmitter release (Brundege and Dunwiddie, 1997). One of these is mediated via a

G-Protein dependent inhibition of N-type Ca++ channels (Mynlieff and Beam, 1994).

This will decrease Ca++-induced transmitter release. Other mechanisms not involving

Ca++ entry from the extracellular space exist (Brundege and Dunwiddie, 1997).

However, it has not been within the scope of the present studies to determine the exact

mechanisms following presynaptic A1-R activation. Nevertheless, we indicate here that

the decreased synaptic activity by adenosine observed in other parts of the brain

(Brundege and Dunwiddie, 1997) is also valid for the medullary central pattern

generator for breathing.


34

Figure 7 Respiration related neurones in the neonatal rat brainstemInspiratory (Insp) neurones receive excitatory synaptic input (EPSPs) and

discharge action potentials during inspiratory C4 ventral root activity (C4). Insp-I

neurones receive EPSPs prior to and after termination of C4 activity whereas Insp-

III neurones are inhibited during both these phases. Biphasic expiratory (Biphasic

E) are characterised by pre- and postinspiratory excitation and inspiratory related

inhibition. Expiratory (Exp) neurones are inhibited by hyperpolarising IPSPs

during the inspiratory phase and either discharge tonically in the expiratory phase

(Exp-i) or receive continued inhibition and discharge primarily in the late

expiratory phase.


35

Figure 8 Irregular C4 respiratory activity (bottom) and discharges of a biphasic

expiratory neurone, discharging before and after C4 and hyperpolarised during C4

inspiratory activity, preparation from new-born pup (2 hours after birth). Note the

regular distance between neuronal discharge burst and how the long postinspiratory

discharges


36

Adenosine and postsynaptic depression of respiration-related neurones

Except for reduced synaptic noise, evident in all neurones, the effects of

adenosinergic agents were different between the major groups of respiration-related

neurones. Expiratory neurones demonstrated a reversible decrease in input resistance,

a depression of action potential discharges and a hyperpolarisation of the membrane

potential during application of R-PIA. Similar responses of input resistance andEm to

R-PIA were apperent after synaptic activity had been blocked by 0.5µM tetrodotoxin

(TTX). As a group, biphasic expiratory (biphasic E) and inspiratory neurones

demonstrated no changes in input resistance or membrane potential during R-PIA

application with our without TTX present. However, whereas the response to

adenosinergic agents was uniform among inspiratory neurones, the response of

biphasic E neurones varied. In some biphasic E neurones the synchronisation between

neurone burst discharges and C4/C5 activity was reversibly disturbed by R-PIA. R-

PIA induced a reversible hyperpolarisation in a third of Biphasic E neurones

examined. Some of these neurones are characterised by a high level of excitatory

(subtreshold) synaptic input and a reduction of ongoing excitatory postsynaptic

potentials could lead to an apparent hyperpolarisation of the membrane potential. The

fact that only one out of 11 Biphasic E neurones demonstrated a change in input

resistance supports this hypothesis. The effects of R-PIA on respiratory activity, input

resistance and membrane potential could be reversed by the A1-receptor antagonist

DPCPX (200 nM).

Our data suggest that the modulation of respiratory output induced by

adenosinergic agents can be explained by: 1) a general decrease in synaptic

transmission between medullary respiration-related neurones mediated by presynaptic

A1-receptors; and 2) an inactivation, via membrane hyperpolarisation, of medullary

expiratory neurones mediated by postsynaptic A1-receptors.

Expiratory neurones are hyperpolarised and inactivated through postsynaptic A1-

receptors. This inactivation of expiratory neurones is similar to what has been

observed in vivo with intracellularly injected adenosine inducing a postsynaptic

membrane hyperpolarisation and inactivation of expiratory neurones (Schmidtet al.,

1995). Alsoin vitro, during hypoxic conditions, the expiratory neurones are reversibly


37

inactivated as demonstrated in the isolated neonatal mice respiratory network (Ramirez

et al., 1998b). Thus, it is plausible that ourin vitro findings could partially explainin

vivo response to hypoxia. Adenosine has been proposed as a link between cellular

energy metabolism and the excitability of neurones (Lloydet al., 1993), since

breakdown of ATP increases substantially during hypoxia and more adenosine is

formed. Adenosine is thus considered as a retaliatory metabolite by which cells

communicate their energy status to surrounding tissues (Newby, 1984).

Inhibition of expiratory neurones does not abolish the respiratory rhythm

As stated above, adenosine causes: 1) a general decrease in synaptic transmission

between medullary respiration-related neurones mediated by presynaptic A1-receptors;

and 2) an inactivation, via membrane hyperpolarisation, of medullary expiratory

neurones mediated by postsynaptic A1-receptors. Inactivation of tonic and late

expiratory neurones only modulates but does not abolish the generation of respiratory

rhythm in vitro. These findings suggests that these neuronesper seare not necessary

for respiratory rhythm generation. This is consistent with the view that respiratory

rhythm is generated primarily by inspiratory-related neurones (Bradleyet al., 1975;

Wyman, 1977; Richter, 1982) and that medullary expiratory neurones are less

important for the rhythmogenesis of respiration. It is contradictory to network models

for the generation of respiratory rhythm in adult mammals, which have phasic and

reciprocal inhibitory interactions between inspiratory and expiratory neurones as a key

feature (Ezure, 1990; Bianchiet al., 1995). However, the respiratory generation

matures during the first two postnatal weeks (Paton and Richter, 1995) and the

neurones in the reduced neonatalin vitro preparation may respond differently than in

adult in vivo systems. Nevertheless, as demonstrated by Richteret al. (Richter et al.,

1993), most expiratory type 2 neurones in the adult cat are inactivated by membrane

hyperpolarisation and stop discharging actionpotentials during hypoxia. As adenosine

levels increase during hypoxia (Winnet al., 1981) a possible explanation would be

that expiratory neuronesin vivo as well asin vitro are not essential for respiratory

rhythm and can be inactivated by hypoxia-induced increased adenosine levels.


38

K+

AdenosineBiphasic

E

Insp

Insp

Type II

Type III

PPPEEERRRIII ---III

Expl

Spinal cord

BiphasicE

InspType I

excitatory

inhibitory

Expt

EEEXXXPPP

IIINNNSSSPPP

Adenosine++Ca

1.

2.

Figure 9 Proposed model for the actions of adenosine A1-receptors (A1-R) in the

respiratory network of neonatal rats. Respiratory neurones are categorised into three

subgroups: inspiratory (INSP), peri-inspiratory (PERI-I) and expiratory (EXP).

1. Presynaptic A1-R decrease neurotransmitter release through inhibiting Ca++-channels

(N-type) (Mynlieff and Beam, 1994).2. Postsynaptic A1-R hyperpolarises and

inactivates expiratory neurones via a decreased K+-channel conductance (Thompsonet

al., 1992). This decreases the overall synaptic transmission in the network without

stopping the respiratory rhythm generation. Identified components of the CPG for

respiration in neonatal ratsin vitro and their connectivity. (Inspiratory neurone (Insp),

late expiratory neurone (Expl), tonic expiratory neurone (Expt), biphasic expiratory

(Biphasic E). The Biphasic E neurone is also characterised as Pre-inspiratory neurone

(Onimaruet al., 1987). (Modified, with kind permission of the authors, after (Onimaru

et al., 1997).


39

DEVELOPMENT OF RESPIRATORY CONTROL

The neuronal network generating respiratory rhythm does not undergo majorchanges in the early perinatal period.

In paper IV we show that an irregular rhythmic respiratory activity is

established in the foetal rat at embryonic day 18. The regularity of respiratory activity,

measured as a decrease in the coefficient of variation (CV) of the interval between C4

burst discharges, increased with postnatal age (P<0.01). Also time of inspiratory

activity in each C4 burst (Ti) increased with postnatal age (ANOVA P<0.05). On E21

a respiratory pattern resembling that after birth had been established with regard to

pontine inhibition, control frequency and Ti. Mean frequency of respiratory activity

did not differ between age groups when pons remained, but was significantly slower

on E18 after ponto-medullary transection. Removal of pons resulted in a significant

increase in frequency (from 2.3+1.4 to 9.1+3.1 burst/min, P<0.0001). The central

pattern generator for respiratory rhythm in medulla oblongata and its inhibition from

pontine structures is thus active on E18. Respiratory rhythmic activity in medulla

oblongata matures pre- and postnatally with regard to both frequency and regularity of

respiratory activity. Pontine inhibition of this respiratory rhythm do not seem to

change from E21 to P3. Our results confirm previous results concerning the

development of respiratory rhythm generation in the foetal and neonatal rat (Di

Pasqualeet al., 1992; Greer , 1992). Previously the inhibition by pons on respiratory

activity in the medulla oblongata has been demonstrated to exist on E20 (Di Pasquale

et al., 1992) but we find it already on E18.

In addition, the depressant effects of an adenosine A1-receptor agonist decrease

between E18 to P3. This parallels the increased regularity of respiratory rhythm and a

possibility could be that a weaker adenosinergic influence on respiration increase its

regularity. However, the increased maturation of dendrites and synaptic connections in

the developing neuronal networks is a more plausible explanation (Bryan and Bryan,

1978). Thus, the decreased role of adenosine in modulating respiration could either be

due to changes in receptor affinity with development (Runoldet al., 1986), or that

more mature neuronal network are less susceptible to the influences of an inhibitory

modulator such as adenosine.


40

The development of respiratory activity and chronic maternal intake of caffeine.

The caffeine treated group differed significantly from control in their basal

respiratory frequency, which was higher than control. Furthermore, the pontine

inhibition of brainstem generated C4 activity was more pronounced in the caffeine

group (MANOVA, repeated measures design; P<0.05) (Paper IV Fig. 2C).

Furthermore, chronic exposure to a low dose of caffeine increased the pontine

inhibition of breathing activity and changed the response to an anoxic challenge

without detectable changes in A1-receptor ontogeny. Furthermore, this pontine

inhibition is dependent on age and is influenced by chronic caffeine intake.

The in vitro findings are strengthened by thein vivo findings of a reduced

gasping frequency during anoxia in the P7 caffeine group. Pons is involved in the

respiratory depression during hypoxia (Okadaet al., 1998). Thus, it is possible that

this reduced gasping frequency reflects an increased hypoxic respiratory depression

mediated by structures in the pons (Okadaet al., 1998).

Even if the levels of adenosine A1-receptors increase with age and high levels are

encountered in pons-medulla in postnatal life (Fig 8) the effects of adenosine on

respiration decrease. It is difficult to quantify the effects of adenosine. The modulation

of synaptic transmission and neuronal membrane properties involve a complex cascade

of mechanisms mediated by several neuromodulators (Lagercrantz, 1987; Feldman,

1990). Changes may be direct by A receptors or indirect effects on other transmitter

systems. Our data indicate that receptor expression is not equivalent to functional

importance. This may be due to that the expression of receptors with increasing

postnatal age are relatively lower in areas and neurones involved in respiratory

regulation compared to surrounding brainstem regions. Post-translatory changes or

changes in network properties may also be involved.

Fig. 10A1-mRNA expression

In a P21 rat


41

GENERAL DISCUSSION

Before birth the isolated respiration-related neuronal networks in the brainstem

generate a similar output as that during the first postnatal days (IV). Thus, changing

respiratory activity at birth from intermittent to continuous breathing does not seem to

depend on major changes in the network.

Birth brings the foetus into contact with a totally different environment than that

in utero. New tactile stimuli, light, lower temperature, removal of the umbilical

circulation, arousal, gas with oxygen and carbon dioxide in the upper and lower

airways, increased pulmonary blood flow, increased oxygen consumption, vagal input

from mechanoreceptors are all involved in the initiation and maintenance of breathing

(Blanco, 1991). The decreased inhibition from neuromodulators such as adenosine and

prostaglandin and increased excitatory afferent input after birth has given rise to the

expression “the inhibited state of the foetus”.

As is apparent from the present studies, the modulatory effects of adenosine on

central respiratory rhythm generation decrease within the first two postnatal days (I,

IV and V). A decreased sensitivity to adenosinergic agents in the neonatal rat after

birth could explain our observations. This concords with earlier findings in neonatal

rabbits (Runoldet al., 1986) and piglets (Elnaziret al., 1996), in which a considerably

more pronounced effect of adenosine agonists on respiration was evident in newborns

(1-3 days) compared with older animals (8 days and 3 weeks). Furthermore, the

present studies indicate that the decreasing sensitivity for adenosine may contribute to

the maintenance of continuous breathing during the first postnatal days. This can be

concluded based on the facts that: 1) adenosine levels ought to decrease as partial

pressure of oxygen in arterial blood rapidly increases after birth; 2), as presented in our

studies (I, IV and V), the effect of A1-R activation on central respiratory activity

decreases during the first two postnatal days.

The instability of breathing in the new-born period has been attributed to the

immaturity of neuronal networks (Bryan and Bryan, 1978). Thus, according to this

view the increased regularity of the respiratory rhythm can be partly explained by the

increased organisation of central neurones as well as the afferent input. The increased

regularity of control respiratory rhythm activities during the perinatal period

investigated in our studies (E18-P7) may be explained by a maturation of the central

neuronal networks. This maturation seems in part to be independent of birth since the


42

increased regularity of central respiratory output in postnatal life has already started in

foetal life (IV). The CV

During pre- and postnatal brain development the brain becomes bigger because

neurones grow in size, and the number of axons and dendrites as well as the extent of

their connections increases (Shatz, 1992). To achieve the precision of the adult pattern,

neuronal function is necessary.

There is a general requirement for neuronal activity to transform immature

neuronal circuits into the organised connections that subserve adequate brain function

(Katz and Shatz., 1996). A key function for neurones is to communicate with other

neurones. “Neurones that fire together wire together while the others are left in the

sink”. Changes in the tonic adenosinergic modulation of synaptic transmission by

caffeine may interfere in the normal development of neuronal circuits. Rat pups whose

mothers drank caffeine had a slightly changed development of respiratory activity and

its control. A speculative explanation to these findings may thus be that the changes

are not due to changes in receptor development but in the development of neuronal

networks.

It may be of functional significance that vital brainstem neurones and their

resulting respiratory outputs remain active, while overall synaptic transmission in the

network is decreased. This will attenuate local neuronal damage induced by hypoxia

and at the same time increase the time for respiration to possibly restore oxygen

saturation to normal levels. Overall, adenosine decreases oxygen consumption and has

neuroprotective effects (Fredholm, 1997) without itself stopping respiratory rhythm

generation. The extensive use of xanthines may thus have unwanted side-effects and

should, may be first be used as a secondary choice of treatment when equivalent

alternatives exists.


43

CONCLUSIONS

• The central respiratory rhythm generation does not undergo major changes in the

perinatal period around birth.

• Adenosine and opioids depress respiration by acting directly on the neuronal

networks in Medulla Oblongata generating respiratory activity. Opioids depress

respiration throughµ- and κ-opioid receptors in the ventral Medulla Oblongata.

The potency ofµ-, but notκ-opioid receptors to depress respiration is temperature

dependent and increases between 22.5-30.5oC.

• The modulatory effect of adenosine and opioids on respiration changes during the

first postnatal days. Whereas adenosinergic depression of respiratory activity is

inversely correlated to perinatal age, the effects of morphine increases with

postnatal age.

• Chronic maternal intake of caffeine during gestation does not affect A1-receptor

development but the development of central respiratory activity and its modulation

by pontine structuresin vitro is altered. Most likely this applies alsoin vivo since

caffeine treated pups, at a postnatal age of 7 days, have a reduced anoxia-induced-

gasping. This indicates that changes in pontine inhibition of respiratory neurones

may have long-term functional consequences. This raises new questions of the

extensive use of caffeine during pregnancy and in treatment of neonates.

• Adenosine depresses respiration through presynaptic adenosine A1-receptor-

mediated inhibition of synaptic transmission in the medulla oblongata.

Furthermore, postsynaptic A1-receptors hyperpolarises and inactivates tonic and

late expiratory neurones but only modulates the generation of respiratory rhythmin

vitro, thus indicating that these neuronesper seare not necessary for respiratory

rhythm generation.


44

ACKNOWLEDGEMENTS

Professor Hugo Lagercrantz, for introducing me to the fascinating field of

developmental physiology and neuroscience, for his great enthusiasm for research and

new ideas, providing laboratory facilities, help with manuscripts and giving me the

possibilities to develop as an independent researcher.

Yuji Yamamoto for introducing me to the isolated respiratory network and Japan

and for his knowledge in neurophysiology.

Professor Sten Lindahl, for never-ending support, advice, bananas and encouragement

as well as crucial help with making some manuscripts readable.

Professor Hiroshi Onimaru, Ikuo Homma and members at their laboratory for giving

me the ability to come to Tokyo and Showa university and hereafter for a welcome

supervision and warmth which made my stay both valuable and memorable.

Koichi Takita, co-author and collaborator for long nights at the laboratory during

experiments and analysis. The smiles and ironic remarks we share

Lie-Qi Tang, Ulrika Åden coauthors and collaborators

Shi-Zeng Yuan and Ronny Wikström for help or dart when needed

Professor Bertil Fredholm for valuable discussions

Dr. Mikael Runold for your helpful advice on life and adenosine, aid with the barometric

method, constructive reading of this thesis and your reflections of life and being such a nice

guy

Professor John Wahren for constructive reading of this thesis.

Jurij Shvarev, Henning Johanssen, Shinhiro T for help in unravelling the mysterious

Torbjörn Hertzberg, Josef Milerad and Hans Holgert for valuable advice

Thomas Ringstedt, Per Uhlen and Ingrid Dahlin for knowing what to do

All colleagues working at the research and neonatal department at Astrid Lindgren

Children’s Hospital


45

Anna Hårdemark for your curiosity, taste for fantasy and bringing life into the

laboratory

Professor Curt von Euler for starting it all

Charin Carlsson and Viveca Karlsson for expert secretarial help, smiles, laughter and

keeping tracks of foundations, grants etc˜.

Professor Sten Grillner and the members at the Department of Neuroscience and

Neurophysiology, for introducing me to the fascinating field of neuroscience and

neuronal networks.

All the staff at the Spinalis unit and members of the Swedish and International

Foundation for Disabled Sailing for giving me the possibility to enjoy sailing and

working in a exciting clinical and sport environment.

Colleagues and friends made during my years in the Doktorandföreningen

Britta and Patrik for being full of laughter, curiosity and being ultimate parents in law

Mom and Dad for bringing me here and opening the world, Nils, Anne and Carl for

help and joys in it.

Joakim, Per, Martin, Niklas for never speaking science

Vivianne, Gittan, Emilie, Christina for always speaking about life

And all of you out there..

Marie the love of my life

Saga Rebecka for being such a wonder

The present study was generously supported by:Swedish Medical Research Council (SMFR 19X-5234, B96-04R-11693 and 14X-

0907), the Swedish Society for Medical Research, Samariten, the Fraenkel and Laerdal

Fund, Wera-Ekstráms Fund for Pediatric Research, Erik and Edith Fernstrám Fund for

Medical Research, Erik Callergrens Memory Foundation, Mia och Erik Myrins

Memory Foundation, Golja and Anna Cederbergs Foundation and Karolinska Institutet

“NO BREATHE, NO LIVE” – The KARATE KID II


46

REFERENCES

Aranda JV and Turmen T (1979), Methylxanthines in apnea of prematurity.Clin Perinatology,

6, 87-108.

Arata A, Onimaru H and Homma I (1990), Respiration-related neurons in the ventral medulla of

newborn rats in vitro.Brain Res Bull, 24, 599-604.

Ball KT, Gunn TR, Gluckmann PD and Power GG (1996), Suppressive action of endogenous

adenosine on ovine fetal nonshivering thermogenesis.Journal of Applied Physiology, 81,2393-2398.

Befort K and Kieffer BL (1997), Structure-activity relationships in the delta opioid receptor.

Pain Reviews, 4, 100-121.

Berne RM (1986), Adenosine: an important physiological regulator.Trends Neuroscience, 1,163-167.

Bianchi AL, Denavit-Saubié M and Champagnat (1995), Central control of breathing in

mammals: neuronal circuitry, membrane properties, and neurotransmitters.Physiological

Rev, 75, 1-45.

Bissonette JM, Hohimer R, Chao CR, Knopp SJ and Notaroberto NF (1990), Theophylline

stimulates fetal breathing movements during hypoxia.Pediatr Res, 28, 83-86.

Blanco C (1991) Role of the brainstem in the changes at birth; initiation of continous breathing

and its maintenance. In:The Fetal and Neonatal Brainstem; developmental and clinical

issues. Edited by M. A. Hanson. Cambridge: University Press, Cambridge, pp. 106-126.

Blanton MG, Lo Turco JJ and Kriegstein, AJ(1989), Whole-cell recordings from neurons in

slices of reptilian and mammalian cerebral cortex.J. Neurosci. Methods, 30, 203-210.

Boddy K, Dawes GS, Fisher RL, Pinter S and Robinson, JS (1974), Foetal respiratory

movements, electrocortical and cardiovascular responses to hypoxaemia.J. Physiol., 243,599-618.

Borday V, Fortin, G and Champagnat J (1997), Early Ontogeny Of Rhythm Generation and

Control Of Breathing.Respiration Physiology, 110,245-249.

Bradley GW, von Euler C, Martilla I and Roos B (1975), A model of the central and reflex

inhibition of inspiration in the cat.Biol. Cybernetics, 19, 105-116.

Brismar H, Asghar M, Carey RM Greengard P and Aperia A (1998), Dopamine-induced

recruitment of dopamine D1 receptors to the plasma membrane.PNAS, 95, 5573-5578.

Brockhaus J, Ballanyi K, Smith JC and Richter DW (1993), Microenvironment of respiratory

neurons in the in vitro brainstem-spinal cord of neonatal rats.J Physiol, 462,421-445.

Brundege JM and Dunwiddie TV (1997), Role of adenosine as a modulator of synaptic activity

in the central nervous system.Advances in Pharmacology, 39, 353-91.

Bryan AC and Bryan MH (1978), Control of respiration in the newborn.Clin Perinatology, 5,269-281.


47

Chernick V and Craig RJ (1982), Naloxone reverses neonatal depression caused by fetal

asphyxia.Science, 216,1252-1253.

Ciruela F, Saura C, Canela EI, Mallol J, Lluis C and Franco R (1997), Ligand-induced

phosphorylation, clustering, and desensitization of A1 adenosine receptors.Molecular

Pharmacology, 52, 788-97.

Cross K and Warner P (1951), The effect of inhalation of high and low oxygen concentration on

the respiration of the newborn infant.J Physiol, 114,283-295.

Darnall RJ (1985), Aminophylline reduces hypoxic ventilatory depression: possible role of

adenosine.Pediatr Res, 19, 706-710.

de Vries JIP, Visser GHA and Prechtl HFR (1982), The emergence of behaviour I. Qualitative

aspects.Early Hum. Dev., 7, 301-322.

Devoe LD, Murray C, Youssif A and Arnaud M (1993), Maternal caffeine consumption and

fetal behavior in normal third-trimester pregnancy.American Journal of Obstetrics &

Gynecology, 168,1105-11.

Di Pasquale E, Monteau R and Hilaire G (1992), In vitro study of central respiratory-like

activity in the fetal rat.Exp Brain res, 89, 459-464.

Dosaka-Akita K Tortella, FC, Holaday JW and Long JB (1993), The kappa opioid agonist U-

50,488H antagonizes respiratory effects of mu opioid receptor agonists in conscious rats.J

Pharmacol Exp Ther, 264,631-637.

Drorbaugh JE and Fenn WD (1955), A barometric method for measuring ventilation in newborn

infants.J Pediatrics,16, 81-87

Drury AN and Szent-Gyrörgyi A (1929), The physiological activity of adenine compounds with

especial reference to their action upon the mammalian heart.J. Physiol, 68, 213-237.

Dunwiddie TV and Fredholm BB (1997), Adenosine regulation of neurotransmitter release. ,kk,kk.

Eldridge FL (1981), Input-output relationships of central neural circuits involved in respiration

in cats.Journal of Physiology, 311,81-95.

Eldridge FL, Millhorn DE and Kiley, JP (1985), Antagonism by theophylline of respiratory

inhibition induced by adenosine.Journal of Applied Physiology, 59 (5),1428-1433.

Eldridge FL, Millhorn DE, Waldrop, TG and Kiley, JP (1983), Mechanism of respiratory effects

of methylxanthines.Respiration Physiology, 53, 239-61.

Elnazir B, Marshall JM and Kumar P (1996), Postnatal development of the pattern of respiratory

and cardiovascular response to systemic hypoxia in the piglet: the roles of adenosine.J

Physiol, 492,573-585.

England SJ (1993) Central effects of hypoxia in the neonate. In:Respiratory control-Central and

Peripheral mechanisms. Edited by Speck DF, Dekin MS, Revelette WR and Frazier . T.

Lexington: The University press of Kentucky, pp. 163-166.

Euler C and Lagercrantz H (1987),Neurobiology of the control of breathing: Raven Press.


48

Ezure K (1990) Synaptic connections between respiratory neurons and considerations on the

genesis of respiratory rhtythm. In:Progress in Neurobiology, Volume 35, pp. 429-450.

Feldman JL (1987) Emergent properties of neuronal mechanisms controling breathing in

mammals. In:Respiratory muscles and their neuromotor control: Alan R. Liss Inc., pp. 3-8.

Feldman JL, Smith JC and Liu G (1991), Respiratory pattern generation in mammals: in vitro en

bloc analyses.Curr Opin Neurobiol, 1, 590-4.

Feldman JL, Smith JC, Ellenberger HH Connelly CA, Liu G, Greer, JJ, Lindsay AD and Otto

MR (1990), Neurogenesis of respiratory rhythm and pattern: emerging concepts.American

Journal of Physiology, 259,879-886.

Flórez J and Hurlé MA (1993) Opioids in respiration and vomiting. In:Hand book of

Experimental pharmacology, Opioids II. Edited by H. A. New York: Springer-Verlag, pp.

263-292.

Fredholm BB (1995), Purinoreceptors in the nervous system.Pharmacology & Toxicology, 76,228-239.

Fredholm BB (1997), Adenosine and neuroprotection. [Review] [130 refs].International Review

of Neurobiology, 40, 259-80.

Garaulet JV, Laorden ML and Milanes MV(1992), Influence of temperature on the effects ofµ-,

δ- andκ-opioid receptor agonists in the guinea-pig ileum myenteric plexus.Eur. J. Pharmac.,

223,19-23.

Greer JJ, Carter JJ and AL-Zubaidy Z (1995), Opioid depression of respiration in neonatal rats.

Journal of Physiology, 485,845-855.

Greer JJ, Smith JC and Feldman JL (1992), Respiratory and locomotor patterns generated in the

fetal rat brain stem-spinal cord in vitro.Journal of Neurophysiology, 67, 996-9.

Grunstein MM, Hazinski, TA and Schlueter MA (1981), Respiratory control during hypoxia in

newborn rabbits: Implied action of endorphins.Journal of Applied Physiology: Respiratory,

Environmental & Exercise Physiology, 51, 122-130.

Guillet R (1990), Neonatal caffeine exposure alters adenosine receptor control of locomotor

activity in the developing rat.Dev. pharmmacol. ther, 15, 94-100.

Guillet R and Dunham L (1995), Neonatal caffeine exposure and seizure susceptibility in adult

rats.Epilepsia, 36, 743-9.

Guntheroth WG and Kawabori I (1975), Hypoxic apnea and gasping.Journal of Clinical

Investigation, 56, 1371-7.

Haddad GG and Mellins RB (1984), Hypoxia and respiratory control in early life. [Review].Ann

Rev Physiol, 46, 629-643.

Hanson M (1996) Neonatal, perinatal and developmental aspects of respiratory control. In:

Pediatrics & Perinatology: The scientific basis. Edited by P. D. Gluckman and M. A.

Heymann. London: Arnold, pp. 836-844.

Hassen AH, Feuerstein G and Faden AI (1984), Kappa opioid receptors modulate

cardiorespiratory function in hindbrain nuclei of rat.J. Neurosci., 4, 2213-2221.


49

Hedner J, Hedner T, Wessberg P, Jonason J and Mueller RA (1985), Central respiratory effects

of adenosine analogues, theophylline and enprofylline. In:Anti-asthma xanthines and

adenosine. Edited by K. E. Andersson and C. G. A. Persson. Copenhagen: Excerpta medica,

pp. 467-471.

Hedner T, Hedner J, Jonason and Wessberg P (1984), Effects of theophylline on adenosine-

induced respiratory depression in the preterm rabbit.Eur J Respir Dis, 65, 153-156.

Hille B (1984),Ionic channels of excitable membranes. Sinauer: Sunderland, M.A.

Holloway Jr WR (1982), Caffeine:Effects of acute and chronic exposure on the behaviour of

neonatal rats.Neurobehv. Toxicol. Teratol., 4, 21-32.

Holloway Jr WRandThor DH (1982), *Caffeine sensitivity in the neonatal rat.Neurobehav.

Toxicol. Teratol, 4, 331-333.

Holloway WRJandThor DH (1982), Caffeine sensitivity in the neonatal rat.Neurobehavioral

Toxicology & Teratology, 4, 331-3.

Irestedt L et al. (1989), Adenosine concentration in umbilical cord blood of newborn infants

after vaginal delivery and cesarean section.Pediatric Research, 26, 106-8.

Isom GE and Elshowihy RM (1982), Naloxone-induced enhancement of carbon dioxide

stimulated respiration,.Life Sci.., 31, 113-118.

Jessell TM and Inversen LL (1977), Opiate analgesics inhibit substance P release from rat

trigeminal nucleus.Nature, 268,549-551.

Johnston BM (1991) Brainstem inhibitory mechanisms in the control of fetal breathing

movements. In:The fetal and neonatal brainstem. Edited by M. Hanson. Cambridge:

Cambridge Uiversity Press, pp. 21-47.

Katz LC and Shatz, JC (1996), Synaptic activity and the construction of cortical circuits.

[Review] [86 refs].Science, 274,1133-8.

Kawai A, Ballantyne D, Muckenhoff K and Scheid P (1996), Chemosensitive medullary

neurones in the brainstem--spinal cord preparation of the neonatal rat.Journal of Physiology,

492,277-292.

Kawai A Okada Y, Muckenhoff K and Scheid P (1995), Theophylline and hypoxic ventilatory

response in the rat isolated brainstem-spinal cord.Resp Physiol, 100,25-32.

Kitterman JA (1996), The effects of mechanical forces on fetal lung growth.Clinics in

Perinatology, 23, 727-40.

Koos BJ and Matsuda K (1990), Fetal breathing, sleep state, and cardiovascular responses to

adenosine in sheep.J Appl Physiol, 68, 489-495.

Kuemmerle JF and Makhlouf GM (1992), Characterization of opioid receptors in intestinal

muscle cells by selective radioligands and receptor protection.Am. J. Physiol., 263,269-276.

Lagercrantz H (1987), Neuromodulators and respiratory control during development.TINS, 10,368-372.


50

Lagercrantz H, Runold M, Yamamoto Y, and Fredholm BB (1986) Adenosine: A putative

mediator of the hypoxic ventilatory response of the neonate. In:Neurobiology of the Control

of Breathing. Edited by H. Euler and H. Lagercrantz. New York: Raven Press, pp. 133-139.

Lagercrantz H, Yamamoto Y, and Fredholm BB, Prabhakar NR and von Euler C (1984),

Adenosine analogues depress ventilation in rabbit neonates. Theophylline stimulation of

respiration via adenosine receptors?Pediatr Res, 18, 387-390.

Lawson EE and Long WA (1983), Central origin of biphasic breathing patterns during hypoxia

in newborns.J. Appl. Physiol., 55, 1299-1304.

Le Gallois (1812),Expériences sur le principe de la vie. Paris: D´Hautel.

Leighton GE, Johnson MA, Meecham KG, Hill RG and Hughes J (1987), Pharmacological

profile of PD117302, a selectiveκ-opioid agonist.Br J Pharmacol, 92, 915-922.

Ling GSF, Simantov R, Clark JA and Pasternak GW (1986), Naloxonazine actions in vivo.Eur.

J. Pharmacol., 129,33-38.

Ling GSF, Spiegel K, Lockhart SH and Pasternak GW (1985), Separation of opioid analgesia

from respiratory depression: evidence for different receptor mechanisms.J. Pharmacol. Exp.

Ther., 232,149-155.

Lloyd HG, Lindström K and Fredholm BB (1993), Intracellular formation and release of

adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion.

Neurochem Int, 23, 173-185.

Lopes JM, Davis GM, Mullahoo K and Aranda JV (1994), Role of adenosine in the hypoxic

ventilatory response of the newborn piglet.Pediatr Pulmonol, 17, 50-55.

Maloney JE, Adamson TM, Brodecky V, Dowling MH and Ritchie BC (1975), Modification of

respiratory center output in the unanesthetised fetal sheep "in utero"J Appl Physiol,39, 552-

558.

Mansour A, Khachaturian H, Lewis ME Akil H and Watson SJ (1988), Anatomy of CNS opioid

receptors.Trends in Neurosci, 11, 308-314.

Miller MJ and Martin RJ (1992) Pathophysiology of apnea of prematurity. In:Fetal and

Neonatal Physiology, Volume 1. Edited by R. F. Polin, WW. Philadelphia: W.B. Saunders

Company, pp. 872-885.

Mitchell GS (1993) Overview: In vitro studies. In:Respiratory control-Cenral and peripheral

mechanisms. Edited by D.F. Speck, M.S. Dekin, W.R. Revelette and D.T. Frazier. Lexington:

Kentucky University press, pp. 30-33.

Mitchell GS and Scheid P (1997), Special Issue - Neural Control Of Breathing - Molecular to

Organismal Perspectives - Foreword.Respiration Physiology, 110,69-70.

Morin SM, Boudinot E, Fournie, Zaluski MC, Champagnat J Roques BP and Denavit-Saubié M

(1992), Control of breathing by endogenous opioid peptides: possible involvement in sudden

infant death syndrome.Neurochem Int, 20, 103-7.

Morton and Garrison (1991),Morton´s Medical Bibliography, Edited by J. M. Norman: Scolar

Press.


51

Moss IR, Denavit-Saubié M, Eldridge FL, Gillis RA, Herkenham M and Lahiri S (1986),

Neuromodulators and transmitters in respiratory control.Federation Proceedings, 45, 2133-

47.

Moss IR, Runold M, Dahlin I, Fredholm BB, Nyberg F and Lagercrantz H (1987), Respiratory

and neuroendocrine responses of piglets to hypoxia during postnatal development.Acta

Physiolog Scandinav, 131,533-541.

Murakoshi T, Suzue T, Tamai S. (1985), A pharmacological study on respiratory rhythm

generation in the in isolated brainstem-spinal cord preparation of the newborn rat.Br. J.

Pharmacology, 86, 95-104.

Mynlieff M and Beam KG (1994), Adenosine acting at an A1 receptor decreases N-type calcium

current in mouse motoneurons.J Neurosci, 14, 3628-3634.

Nehlig A and Debry G (1994), Potential teratogenic and neurodevelopmental consequences of

coffe and caffeine exposure: a review on human and animal data.Neurotoxicology and

teratology, 16, 531-543.

Neubauer JA, Melton JE and Edelman NH (1990), Modulation of respiration during hypoxia.J

Appl Physiol, 68, 441-451.

Newby AC (1984), Adenosine and the concept of "retaliatory metabolites".Trends Biochem Sci,

9, 42-49.

Neylon M and Marshall JM (1991), The role of adenosine in the respiratory and cardiovascular

response to systemic hypoxia in the rat.J Physiol, 440,529-545.

Okada Y, Kawai A, Muckenhoff K and Scheid P (1998), Role of the pons in hypoxic respiratory

depression in the neonatal rat.Respiration Physiology, 111,55-63.

Okada Y, Muckenhoff K, Holterman G, Acker H and Scheid P (1993), Depth profiles of pH and

and PO2 in the isolated brainstem spinal cord of the neonatal rat.Respir Physiol, 93, 315-326.

Olson EB Jr (1987), Naloxone accelerates the rate of ventilatory acclimatization to hypoxia in

awake rats.Life Sci., 41, 161-167.

Onimaru H (1995a), Studies of the respiratory center using isolated brainstem-spinal cord

preparations. [Review] [79 refs].Neuroscience Research, 21, 183-90.

Onimaru H, Arata A and Homma I (1987), Localization of respiratory rhythm-generating

neurons in the medulla of brainstem-spinal cord preparations from newborn rats.

Neuroscience Letters, 78, 151-5.

Onimaru H, Arata A and Homma I (1988), Primary respiratory rhythm generator in the medulla

of brainstem-spinal cord preparation from newborn rat.Brain Research, 445,314-24.

Onimaru H, Arata A and Homma I (1990), Inhibitory synaptic inputs to the respiratory rhythm

generator in the medulla isolated from newborn rats.Pflugers Arch, 417,425-432.

Onimaru H, Arata A and Homma I (1997), Neuronal mechanisms of respiratory rhythm

generation: an approach using in vitro preparation.Japanese Journal of Physiology, 47, 385-

403.


52

Onimaru H, Ballanyi K and Richter DW (1996), Calcium-dependent responses in neurons of the

isolated respiratory network of newborn rats.J Physiol (London), 491,677-95.

Onimaru H and Homma I (1992), Whole cell recordings from respiratory neurons in the medulla

of brainstem-spinal cord preparations isolated from newborn rats.European journal of

physiol., 420,399-406.

Onimaru H, Arata A and Homma I (1995b), Intrinsic burst generation of preinspiratory neurons

in the medulla of brainstem-spinal cord preparations isolated from newborn rats.

Experimental Brain Research, 106,57-68.

Orlowski JP (1986), Cerebrospinal fluid endorphins and the infant apnea syndrome.Pediatrics,

78, 233-237.

Pasternak GW, Childers SR and Snyder SH (1980), Opiate analgesia: evidence for mediation by

a subpopulation of opiate receptors.Science, 208,514-516.

Paton JF and Richter DW (1995), Maturational changes in the respiratory rhythm generator of

the mouse.Pflugers Archiv European Journal of Physiology, 430,115-24.

Pierrefiche O, Schwarzacher SW Bischoff AM and Richter DW (1998), Blockade of synaptic

inhibition within the pre-Botzinger complex in the cat suppresses respiratory rhythm

generation in vivo.Journal of Physiology, 509,245-54.

Puig MM, Warner W Tang CK, Laorden ML and Turndorf H (1987), Effects of temperature on

the interaction of morphine with opioid receptors,.Br. J. Anaesth., 59, 1459-1464.

Ramirez JM, Quellmalz UJA, Wilken B and Richter DW (1998a), Developmental changes in the

hypoxic response of hypoglossus respiratory motor output in vitro.Journal of

Neurophysiology, 78, 383-392.

Ramirez JM, Quellmalz UJA, Wilken B and Richter DW (1998b), The hypoxic response of

neurones within the in vitro mammalian respiratory network.Journal of Physiology, 507,571-582.

Ramirez JM, Schwarzacher SW, Pierrefiche O, Schwarzacher SW and Richter DW (1998c),

Selective lesioning of the cat pre-Bötzinger complex in vivo eliminates breathing but not

gasping.Journal of Physiology, 507,895-907.

Ramirez JM, , Telgkamp P Elsen FP, Quellmalz UJA, and Richter DW (1997), Respiratory

Rhythm Generation In Mammals - Synaptic and Membrane Properties.Respiration

Physiology, 110,71-85.

Rehan V, Haider, AZ, Alvaro RE, Nowaczyk B, Cates DB, Kwiatkowski K and Rigatto H

(1996), The biphasic ventilatory response to hypoxia in preterm infants is not due to a

decrease in metabolism.Pediatric Pulmonology, 22, 287-294.

Rekling J, Champagnat J and Denavit-Saubié M (1996), Thyrotropin-releasing hormone (TRH)

depolarizes a subset of inspiratory neurons in the newborn mouse brain stem in vitro.Journal

of Neurophysiology, 75, 811-9.


53

Rekling JC and Feldman JL (1998), Prebötzinger complex and pacemaker neurons -

hypothesized site and kernel for respiratory rhythm generation [Review].Annual Review of

Physiology, 60, 385-405.

Richter DW (1982), Generation and maintenance of the respiratory rhythm.Journal of

Experimental Biology, 100,93-107.

Richter DW (1992), Mechanisms of respiratory rhythm generation. [Review].Current Opinion

in Neurobiology, 2, 788-93.

Richter DW, Bischoff A, Anders K, Bellingham M and Windhorst U (1993) Modulation of

respiratory patterns during hypoxia. In:Respiratory Control Central and Peripheral

mechanisms. Edited by D. F. Speck, M. S. Dekin, W. R. Revelette and D. T. Frazier.

Lexington: The university Press of Kentucky, pp. 21-28.

Rigatto H, Brady JP and Chir B (1972), Periodic breathing and apnea in preterm infants. II.

Hypoxia as a primary event.Pediatrics, 50, 219-28.

Runold M, Lagercrantz H and Fredholm BB (1986), Ventilatory effect of an adenosine analogue

in unanesthetized rabbits during development.J Appl Physiol, 61, 255-259.

Runold M, Lagercrantz H, Prabhakar NR and Fredholm BB (1989), Role of adenosine in

hypoxic ventilatory depression.J Appl Physiol, 67, 541-546.

Sawynok J (1998), Adenosine receptor activation and nociception.Eur J Pharm, 317,1-11.

Sawynok J, Sweeney MI and White TD (1989), Adenosine release may mediate spinal analgesia

by morphine [see comments]. [Review].Trends in Pharmacological Sciences, 10, 186-9.

Schmidt C et al. (1995), Adenosinergic modulation of respiratory neurones and hypoxic

responses in the anaesthetized cat.J Physiol, 483,769-781.

Schwieler (1968),Acta Phys Scand Suppl, 304,1-123.

Shao XM and Feldman JL (1997), Respiratory Rhythm Generation and Synaptic Inhibition Of

Expiratory Neurons In Pre-Bötzinger Complex - Differential Roles Of Glycinergic and

Gabaergic Neural Transmission.Journal of Neurophysiology, 77, 1853-1860.

Shatz C (1992), The developing Brain.Scientific American, 267,35-41.

Shook JE, Watkins WD and Camporesi EM (1990), Differential roles of opioid receptors in

respiration, respiratory disease, and opiate-induced respiratory depression.Am. Rev. Respir.

Dis., 142,895-909.

Sinton CM, Valatay JL and Jouvet M (1981), Gestational caffiene modifies offspring behaviour

in mice.Psycopharmacology, 75, 69-74.

Smith JC, Ellenberger HH, Ballanyi K, Richter DW and Feldman, JL (1991), Pre-Bötzinger

complex: a brainstem region that may generate respiratory rhythm in mammals.Science, 254,726-729.

Smith JC, Greer JJ, Liu GS and Feldman, JL (1990), Neural mechanisms generating respiratory

pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and

medullary neuron activity.J Neurophysiol, 64, 1149-1169.


54

Snyder SH (1985), Adenosine as a neuromodulator. [Review].Annual Review of Neuroscience,

8, 103-24.

Sobotka TJ, Spaid SJ and Brodie RE (1979), Neurobehaviuoral teratology of caffeine exposure

in rats.Neurotoxicology, 1, 403-416.

Sollevi A (1997), Adenosine for pain control.Acta Anaesthesiologica Scandinavica.

Supplementum., 110,135-6.

Speck DF, Dekin DF, Revelette WR and Frazier DT (1993), Respiratory Control: Central and

Peripheral Mechanisms. Lexington: The University Press of Kentucky, pp. 238.

Steinbrook RA, Feldman HA, Fencl V, Forte VA Jr, Gobel RA, Leith, DE and Weinberger SE

(1984), Naloxone does not affect ventilatory responses to hypoxia and hypercapnia in rats.

Life Science, 34, 881-887.

Suzue T (1984), Respiratory rhythm generation in the in vitro brainstem-spinal cord preparation

of the neonatal rat.J Physiol, 354,173-183.

Taguchi Ket al. (1993), Effects of morphine on release of acetylcholine in the rat striatum: an in

vivo microdialysis study.Naunyn-Schmiedebergs Arch Pharmacol, 347, 9-13.

Thomas T, Elnazir, BK and Marshall JM 1994), Differentiation of the peripherally mediated

from the centrally mediated influences of adenosine in the rat during systemic hypoxia.Exper

Physiol, 79, 809-822.

Thomas T and Soyer KM (1996), The role of adenosine receptors in the rostral entrolateal

medulainthe cardiovascular response to defence area stimulaton in the rat.Exp Physiol, 81,67-77.

Thompson SM, Haas HL and Gahwiler BH (1992), Comparison of the actions of adenosine at

pre- and postsynaptic receptors in the rat hippocampus in vitro.J Physiol Lond, 451,347-63.

Traynor JR (1996), Theµ-opioid receptor.Pain Reviews, 3, 221-248.

Vitez TS, White TS and Eger El il (1974), Effects of hypothermia on halothane MAC and

isoflurane MAC in the rat.Anesthesiology, 41, 80-1.

Voipio J and Ballanyi K (1997), Interstitial P-Co2 and Ph, and Their Role As Chemostimulants

In the Isolated Respiratory Network Of Neonatal Rats.Journal of Physiology London, 499,527-542.

von Euler C (1980), Central pattern generation during breathing.TINS, 3, November, 275-277.

von Euler C (1991) Neuronal organization and rhythm generation. In:The Lung. Edited by R. G.

Crystal and J. B. West. New York: Raven Press, Ltd., pp. 1-12.

Waites BA, Ackland GL, Noble R and Hanson MA (1996), Red nucleus lesions abolish the

biphasic respiratory response to isocapnic hypoxia in decerebrate young rabbits.Journal of

Physiology, 495,217-25.

Winn HR, Rubio R and Berne RM (1981), Brain adenosine concentration during hypoxia in rats.

Am J Physiol, 241,235-242.

Wyman RJ (1977), Neuronal generation of the breathing rhythm.Ann. Rev. Phys., 39, 417-448.


55

Xia Y and Haddad GG (1991), Ontogeny and distribution of opioid receptors in the rat

brainstem.Brain Research, 549,181-193.

Yeadon M and Kitchen I (1989), Opioids and respiratory depression.Prog. Neurobiol., 33, 1-16.

Yeadon M and Kitchen I (1990), Multiple opioid receptors mediate the respiratory depressant of

fentanyl-like drugs in the rat.Gen. Pharmacol., 21, 655-664.

Documents

RESPIRATORY ACTIVITY IN MEDULLA OBLONGATA and its modulation by