Jean Champagnat

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 andNT4, revealed that survival of many NPG afferents is proportional to the number of functional BDNF alleles, whereas only onefunctional 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.

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.

Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex.

The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

Catecholaminergic depressant effects on bulbar respiratory mechanisms

On the basis of histochemical and pharmacological studies, catecholamines have been implicated in central mechanisms controlling respiration. This hypothesis was tested in iontophoretic studies on neurones located in bulbar respiratory centres. Adrenaline and noradrenaline had a predominantly depressant effect on respiratory as well as on closely situated non-respiratory units. These depressions were mimicked by the application of isoproterenol and clonidine; acetylcholine and serotonin had inconsistent effects on these neurones. In control experiments, microinjections, using a Hamilton syringe, were made in the area of bulbar respiratory centres: noradrenaline, but not serotonin, depressed the central respiratory activity reflected in the phrenic nerve discharge. These results suggest that specific adrenergic and noradrenergic depressant mechanisms could affect both respiratory and other physiological centres at the bulbar level.

Involvement of amino acids in periodic inhibitions of bulbar respiratory neurones

As previously demonstrated, spontaneously firing bulbar inspiratory neurones are periodically inhibited either at the beginning of, or throughout expiration, while bulbar expiratory neurones are inhibited during inspiration. The aim of the present study was to test the hypothesis that amino acids act as transmitters of these periodic inhibitions. The study was performed using iontophoretic applications of drugs on bulbar respiratory neurones. On these neurones GABA and glycine-sensitive sites were identified and differentiated on the basis of the actions of agonist (muscimol) or antagonists (bicuculline, picrotoxin and strychnine). Using competitive antagonists (nipecotic acid, beta-alanine) mechanisms responsible for GABA uptake were found in the close vicinity of respiratory-related neurones. Some but not all types of periodic inhibition were found to be reduced following application of GABA or glycine antagonists. Strychnine was found to reduce periodic inhibitions occurring at the beginning of expiration in inspiratory neurones. GABA antagonists had an effect on those periodic depressions which were prolonged throughout expiration. A different and complementary role of glycine-like and GABA-like systems in central respiratory mechanisms is proposed.

Emergence of the Pre-Bötzinger Respiratory Rhythm Generator in the Mouse Embryo

To obtain insights into the emergence of rhythmogenic circuits supporting respiration, we monitored spontaneous activities in isolated brainstem and medullary transverse slice preparations of mouse embryos, combining electrophysiological and calcium imaging techniques. At embryonic day 15 (E15), in a restricted region ventral to the nucleus ambiguus, we observed the onset of a sustained high-frequency (HF) respiratory-like activity in addition to a preexisting low-frequency activity having a distinct initiation site, spatial extension, and susceptibility to gap junction blockers. At the time of its onset, the HF generator starts to express the neurokinin 1 receptor, is connected bilaterally, requires active AMPA/kainate glutamatergic synapses, and is modulated by substance P and the μ-opioid agonist d-Ala2-N-Me-Phe4-Glycol5-enkephalin. We conclude that a rhythm generator sharing the properties of the neonatal pre-Bötzinger complex becomes active during E15 in mice.

Primordial respiratory-like rhythm generation in the vertebrate embryo

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated centrally, within neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the best understood with respect to the cellular and molecular mechanisms that underlie its development. Examination of hindbrain activities in the chick embryo has revealed that the central rhythm generator is active before fetal maturation and conforms to the rhombomeric organization of the embryonic hindbrain. Inactivation of genes required for the normal formation of rhombomeres in mice leads to perturbations of the reticular formation that affect respiration after birth and compromise the probability of survival. From studies of hindbrain development we might gain an understanding of how genes govern the early embryonic development of neuronal networks and how this might specify patterns of motor activities operating throughout life.

NMDA and non-NMDA receptors may play distinct roles in timing mechanisms and transmission in the feline respiratory network

1. Activation of N‐methyl‐D‐aspartate (NMDA) glutamate receptors in the brainstem network of respiratory neurones is required to terminate inspiration in the absence of lung afferents, but it is not required in the inspiratory motor act of lung inflation. In the present study we examined the involvement of non‐NMDA ionotropic glutamate receptors in these two mechanisms in the adult mammal. 2. Adult cats were either decerebrated or anaesthetized with sodium pentobarbitone, paralysed and ventilated. Inspiratory motor output was recorded from the phrenic nerve and central respiratory activity from neurones in the bulbar ventral respiratory group. 3. In decerebrate vagotomized cats, ionophoretic application of 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoylbenzo(F)quinoxaline (NBQX) onto single respiratory neurones decreased their spontaneous discharge rate and abolished the excitatory effect of exogenously applied (RS) alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (AMPA) but not NMDA. 4. In these animals, intravenous infusion (12 mg kg‐1) of the non‐NMDA receptor blockers GYKI 52466 (1‐(4‐aminophenyl)‐4‐methyl‐7,8‐methylene‐dioxy‐5‐H‐2,3‐benzodi aze pine) or NBQX: (1) decreased (in 10/15 cats) or abolished (in 5/15 cats) the inspiratory‐related discharge of the phrenic nerve; (2) did not prolong the inspiratory phase; (3) reduced or abolished the spontaneous discharge of respiratory neurones; and (4) profoundly decreased the excitatory effects of AMPA but not NMDA ionophoresed onto these neurones. When both the phrenic nerve and the recorded respiratory neurone were silenced, neuronal excitation by ionophoretic application of NMDA first revealed a subthreshold respiratory modulation without lengthening of the inspiratory phase, then respiratory modulation became undetectable. 5. Additional blockade of NMDA receptors by a small dose (0.15 mg kg‐1) of dizocilpine (MK‐801), abolished the phrenic nerve activity which persisted after NBQX (apnoea), but the discharge or the subthreshold modulation of the bulbar respiratory neurones showed a lengthening of the inspiratory phase (apneusis). 6. Elevation of FA,CO2 increased or re‐established phrenic nerve discharges after blockade of non‐NMDA receptors or of both NMDA and non‐NMDA receptors. 7. Small doses of NBQX or GYKI 52466 induced apnoea in five of five cats anaesthetized with sodium pentobarbitone. 8. In decerebrate animals with intact vagi, GYKI 52466 and NBQX depressed the Hering‐Breuer expiratory‐lengthening reflex. 9. The results suggest that: (1) there is a specialization of different classes of glutamate receptors participating in timing mechanisms and transmission within the mammalian respiratory network. Neural transmission predominantly involves activation of non‐NMDA receptors, acting in synergy with NMDA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Rhythm generation in the segmented hindbrain of chick embryos.

1. The embryonic hindbrain of chick is segmented until stage 24, when it starts to generate rhythmic activities in cranial nerves. In order to recognize a possible influence of segmentation on the organization of neuronal systems generating motor rhythms, the activity of trigeminal, facial, glossopharyngeal, vagal and hypoglossal nerves was studied during embryonic stages 24‐36, by simultaneous recording of different cranial nerves in an isolated, superfused chick hindbrain preparation. 2. Highly correlated recurrent episodes of cyclical burst discharges occurred in all nerves studied (correlation coefficients, 0.8 +/‐ 0.1) throughout stages 24‐36. 3. Such coactivation is unlikely to be due to monosynaptic connections between widely divergent premotor neurons and motoneurons, or between motoneurons themselves, because no short‐term correlation was apparent in the millisecond range between activities of different motor nerves. 4. Complete transverse or midsagittal sectioning of the hindbrain disrupted coactivation of nerves located at distinct rostrocaudal levels or occupying an ipsi‐ or contralateral position, respectively, while sparing the ability of individual nerves to generate rhythmic activity. Each hindbrain segment thus contains bilaterally the motor nuclei together with their own rhythm generator. Coactivation of motor patterns appears to result from intersegmental and cross‐median connections between these rhythm generators. 5. The results are in keeping with the hypothesis of a segmental organization of the primordial hindbrain rhythm generator and give further support to the early determination of both the anatomical and the functional fate of neurons in this region of the vertebrate central nervous system.

Spontaneous synaptic activities in rat nucleus tractus solitarius neurons in vitro: evidence for re-excitatory processing.

The pattern of synaptic interactions between neurons of the nucleus tractus solitarius (NTS) has been analyzed using whole cell recording in rat brainstem slices. Following tractus solitarius (TS) stimulation 15/55 neurons presented a prolonged (up to 300 ms) increased excitability (PIE neurons) and 40/55 neurons presented a prolonged (up to 200 ms) reduced excitability (PRE neurons). In the absence of afferent sensory input all neurons showed spontaneous synaptic activity. Ongoing synaptic activity in PIE cells was glutamatergic and characterized by the absence of detectable inhibitory potentials while in PRE cells it was 90% GABAergic and 10% glutamatergic. Glutamatergic synaptic currents in PIE cells and GABAergic synaptic currents in PRE were studied using probability density and intensity functions. Distribution of time intervals between synaptic events indicated the latter were generated, in both PIE and PRE cells, by two simultaneous processes: (1) a close to Poisson process generating independent events; and (2) a subsidiary re-excitatory process generating synaptic events separated by intervals shorter than 20 ms. Blockade of glutamatergic transmission by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) or blockade of action potentials by tetrodotoxin (TTX; 1 microM) suppressed the subsidiary process. In conclusion, we propose that PIE cells (1) form a re-excitatory network contributing to generation of excitatory activity in the NTS and (2) are located presynaptically with respect to PRE cells.