Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.

Early organization of the vertebrate brainstem is characterized by cellular segmentation into compartments, the rhombomeres, which follow a metameric pattern of neuronal development. Expression of the homeobox genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they play an important role in the establishment of rhombomere-specific neuronal patterns. However, segmentation is a transient feature, and a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages. Thus, it is not clear whether the early rhombomeric pattern of Hox expression has any influence on the establishment of the neuronal circuitry of the mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of mouse Hoxa1(-/-) mutants has focused on early alterations of hindbrain segmentation and patterning. Here, we show that ectopic neuronal groups in the hindbrain of Hoxa1(-/-) mice establish a supernumerary neuronal circuit that escapes apoptosis and becomes functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2 identity, and integrates the rhythm-generating network controlling respiration at birth. This is the first demonstration that changes in Hox expression patterns allow the selection of novel neuronal circuits regulating vital adaptive behaviors. The implications for the evolution of brainstem neural networks are discussed.

evx1 transcription in bony fin rays segment boundaries leads to a reiterated pattern during zebrafish fin development and regeneration.

The dermoskeleton of zebrafish fins is composed of actinotrichia and segmented bony rays, or lepidotrichia, which grow by successive addition of distal segments. The present study shows that evx1, a new zebrafish even-skipped related gene (Thaëron et al., 2000) displays during bony ray morphogenesis, a unique repetitive expression pattern along the proximodistal axis of the fin. Whole-mount in situ hybridization performed on larvae and adult regenerating fins show that evx1 signal appears as parallel dash lines crossing the width of each developing and regenerating rays, in a ladder-like fashion. Cytological studies show that a subpopulation of bone forming cells (scleroblasts) expresses evx1 at the level of the joint between two adjacent segments except in the apical part of the differentiating ray where evx1 expression precedes the formation of the joint. This distal transcription is turned on again only when the latest differentiating segment reached its final size and might label the putative next segment boundary. This suggests the existence of a molecular mechanism controlling the periodic expression of evx1 which could be involved in the establishment of segment boundaries during fin ray morphogenesis, and could play a key role during dermal skeleton patterning.

Zebrafish evx1 is dynamically expressed during embryogenesis in subsets of interneurones, posterior gut and urogenital system.

The even-skipped-related homeobox genes (evx) are widely distributed through animal kingdom and are thought to play key role in posterior body patterning and neurogenesis. We have cloned and analyzed the expression of evx1 in zebrafish (see also Borday et al. (Dev. Dyn. 220 (2001) in press) which displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurones prior to their axonal outgrowth, identified as primary interneurones and a subset of Pax2.1(+) commissural interneurones. In the hindbrain, evx1 is expressed in reticulospinal interneurones of rhombomeres 5 and 6 as well as in rhombomere 7 interneurones. The latest emerging evx1(+) interneurones in the hindbrain correspond to commissural interneurones. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the uro-genital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast other vertebrates, evx1displays no early nor caudal expression in zebrafish.

Pontine and medullary control of the respiratory activity in the trigeminal and facial nerves of the newborn mouse: an in vitro study.

In vitro, the respiratory activity in rodents is characterized by: (i) the rapidly peaking, slowly decrementing pattern of spontaneous and rhythmic active phases recorded from the motor rootlets, and (ii) the specific location of their rhythmic generator in the ventrolateral medulla. The aim of the present study was to assess whether the trigeminal and facial motor rootlets still exhibit respiratory activity in the absence of peripheral and higher cerebral structures, and to compare the onset of their active phases with that of other respiratory rootlets, using the in vitro isolated brainstem--spinal cord preparation of the newborn mouse and rat. Spontaneous rhythmic activity was recorded from the trigeminal and facial rootlets. It was regular and synchronized bilaterally and ipsilaterally with the hypoglossal or cervical C1-C6 rootlets. Brainstem transection experiments demonstrated that for both the trigeminal and facial rootlets, the spontaneous rhythmic activity originates from the medulla, in a region consistent with the pre-Bötzinger complex and the rostral ventrolateral medulla. The pattern of the respiratory motor activity recorded from the trigeminal and facial rootlets was identical to the pattern recorded from the hypoglossal and cervical C1-C6 rootlets with rapidly peaking, slowly decrementing characteristics. The duration of the ascending part and the total duration of their active phases were similar. The onset of the active phases of the phrenic rootlets was delayed compared with that of the trigeminal, facial and hypoglossal rootlets. However, no difference in the onsets of the active phases of the cranial rootlets could be observed. Removal of the rostral pons suppressed the delay in onset of the active phases of the phrenic rootlets. Our findings show that: (i) rhythmic activities of the trigeminal and facial rootlets are preserved in absence of control by peripheral or high cerebral structures; (ii) the pattern and the location of the rhythmic generator for these activities are of the respiratory type; and (iii) the rostral pons is responsible for a delay in the onset of the active phases of the phrenic rootlets compared with that of the trigeminal, facial and hypoglossal rootlets.

Respiratory effects of glutamate receptor antagonists in neonate and adult mammals.

We determined the conditions (immaturity, species, anesthesia, receptor blockade selectivity) under which glutamate receptor blockade produces respiratory depression in mammals. In unrestrained 0- to 2-day-old neonate and adult mice and cats, ventilation was measured by the barometric method, before and after separate or sequential administration of a non-NMDA receptor antagonist, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline, 2-200 mg kg(-1) in mice, 10-40 mg kg(-1) in cats), and a NMDA receptor antagonist, dizocilpine (3 mg kg(-1) in mice, 0.15-1.0 mg kg(-1) in cats). NBQX or dizocilpine alone did not decrease ventilation in awake adults, but NBQX strongly depressed ventilation in neonate awake mice and in adult anesthetized animals. Given together, dizocilpine and NBQX always profoundly depressed ventilation by producing a lethal apnea in neonate mice, and an apneustic pattern of breathing in adults of both species and in neonate cats. We conclude that blockade of either NMDA or non-NMDA receptors is innocuous in awake adults. The factors which may potentiate respiratory depression are (1) anesthesia, (2) immaturity, and (3) combined blockade of both receptors types. The mechanism of depression is species-dependent and age-dependent.

Early ontogeny of rhythm generation and control of breathing.

The ability of central networks to produce rhythmic motor behaviours linked to the respiratory function, is a remarkably conserved property of the brainstem reticular formation in vertebrates. Conserved cellular and molecular mechanisms also underlie the early embryonic development of the brainstem, leading to a segmented rhombencephalon in all vertebrates. We have proposed that the neural network that controls breathing after birth, derives from a primordial rhythmic network first active in the segmented hindbrain of the embryo. Observations on transgenic mice support this hypothesis: homozygous inactivation of Krox-20, a gene governing segmentation, leads to a lower-than-normal respiratory frequency (fR), despite fetal maturation of the respiratory network and functional compensatory control after birth.

A ventral pontine pathway promotes rhythmic activity in the medulla of neonate mice.

We have developed in new-born mice a ventral tilted-horizontal slice preparation for pontine stimulation and recording of spontaneous respiratory-like rhythmic trains of glutamatergic excitatory postsynaptic potentials (EPSPs) in medullary neurons. Electrical stimulations (10-50 Hz for 100-500 ms) of the caudal pontine reticular formation triggered a burst of EPSPs, recycling of the rhythmic activity and persistent increase of the rhythmic behaviour. These results identify a ventral pontine pathway that promotes rhythm generating mechanisms in the medulla and probably derives from a population of lateral reticular neurons identified in the embryonic hindbrain and eliminated after inactivation of the early developmental gene Krox-20.

Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice.

We have shown previously that the inactivation of the zinc finger gene Krox-20 affects hindbrain segmentation, resulting in the elimination of rhombomeres 3 and 5. We demonstrate here that Krox-20 homozygous mutant mice exhibit abnormally slow respiratory and jaw opening rhythms, indicating that a modification of hindbrain segmentation influences the function of neuronal networks after birth. Central neuronal networks that control respiratory frequency are made predominantly depressant by the elimination of a previously undescribed rhythm-promoting system. Recordings of rhythmic activity from the isolated hindbrain following progressive tissue transections indicate that the reorganization takes place in the caudal pontine reticular formation. The newborn (PO) Krox-20-/- mice, in which apneas are ten times longer than in wild-type animals, may be a valuable model for the study of life-threatening apneas during early infancy.

Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4) act via the TrkB receptor and support survival of primary somatic and visceral sensory neurons. The major visceral sensory population, the nodose-petrosal ganglion complex (NPG), requires BDNF and NT4 for survival of a full complement of neurons, providing a unique opportunity to compare gene dosage effects between the two TrkB ligands and to explore the possibility that one ligand can compensate for loss of the other. Analysis of newborn transgenic mice lacking BDNF or NT4, or BDNF and NT4, revealed that survival of many NPG afferents is proportional to the number of functional BDNF alleles, whereas only one functional NT4 allele is required to support survival of all NT4-dependent neurons. In addition, subpopulation analysis revealed that BDNF and NT4 can compensate for the loss of the other to support a subset of dopaminergic ganglion cells. Together, these data demonstrate that the pattern of neuronal dependencies on BDNF and NT4 in vivo is far more heterogeneous than predicted from previous studies in culture. Moreover, BDNF knockout animals lack a subset of afferents involved in ventilatory control and exhibit severe respiratory abnormalities characterized by depressed and irregular breathing and reduced chemosensory drive. BDNF is therefore required for expression of normal respiratory behavior in newborn animals.