Fabien Tell

Postnatal changes in electrophysiological properties of rat nucleus tractus solitarii neurons.

Whole‐cell recordings in brainstem slices revealed postnatal changes in passive and firing properties in the rat caudal nucleus tractus solitarii (cNTS) neurons. Membrane potential, threshold for Na+ spike and degree of sag were unchanged during development. In the adult, the rheobase was twice that found at birth. The input resistance decreased over the period studied, while time constants declined markedly after the third postnatal week. At all postnatal ages, Na+‐dependent action potentials (APs) were elicited in response to depolarization. Nevertheless, AP duration gradually decreased by 40% over the developmental period studied. Spike amplitude was smaller at birth than at any other ages and reached a peak two weeks after birth. At all ages, Na+‐dependent APs were blocked by application of tetrodotoxin. Full APs were replaced by an initial slow oscillation in young cells and by oscillations in older cells. The TTX‐resistant oscillations were altered by cobalt (2 mM) and cadmium (100 μM). The spike afterhyperpolarization (AHP) was not altered during development, but was observed in less neurons in adult cells when measured at a holding potential of ‐60 mV. Neurons were subdivided into one of three classes based on their responses to a hyperpolarizing prepulse: 1) post‐inhibitory rebound (PIR) cells, 2) delayed excitation (DE) cells and 3) NON cells expressing neither PIR nor DE. The relative proportions of different cell types varied with age. The mean maximum duration of DE increased three times. Voltage‐clamp experiments revealed that the DE was due to the activation of an A‐current. In addition, a three‐fold increase in its inactivation rate was observed postnatally. The physiological significance of these results is discussed.

PERINATAL DEVELOPMENTAL CHANGES IN RESPIRATORY ACTIVITY OF MEDULLARY AND SPINAL NEURONS : AN IN VITRO STUDY ON FETAL AND NEWBORN RATS

Experiments were performed in vitro on fetal and newborn rat brainstem-spinal cord preparations to analyse the perinatal developmental changes in inspiratory motor output. The amplitude of the inspiratory bursts of the whole C4 ventral root (global extracellular recording), the firing patterns of 80 medullary inspiratory neurons (unitary extracellular recording) and the firing and membrane properties of 71 respiratory neurons in the C4 ventral horn (whole-cell recording) were analysed at embryonic day 18 (E18), 21 (E21) and post natal days 0 to 3 (P0-3). At E18, the amplitude of the C4 bursts was weak and variable from one respiratory cycle to the next, as well as the discharge pattern of most of the medullary inspiratory neurons. C4 motoneurons were immature, very excitable and displaying variable inspiratory discharges, but already able to deliver sustained bursts of potentials when depolarised. At E21 and P0-3, the amplitude of the C4 bursts was increased and stable, most of the medullary inspiratory neurons already were able to generate a stable firing pattern and C4 motoneurons showed maturational changes in terms of the resting potential, spike amplitude and input membrane resistance. This work suggests that the short period extending from E18 to E21 is a critical maturational period for the medullary respiratory network which becomes able to elaborate a stable respiratory motor output.

Cellular and synaptic effect of substance P on neonatal phrenic motoneurons

Experiments were carried out on the in vitro brainstem–spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current‐clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency–intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP‐mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin‐containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage‐clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4‐aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M‐type potassium current but only a current showing no voltage sensitivity over the range −100 to 0 mV, reversing near the expectedEK+, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.

Activation of N-methyl-d-aspartate Receptors Induces Endogenous Rhythmic Bursting Activities in Nucleus Tractus Solitarii Neurons: An Intracellular Study on Adult Rat Brainstem Slices.

A brainstem slice preparation and intracellular recording techniques were used to examine the effects of N‐methyl‐D‐aspartate (NMDA) application on neurons within the swallowing area of the nucleus tractus solitarii (NTS). According to their cellular properties, NTS neurons were classified into type I and type II neurons. The most striking difference was the occurrence of delayed excitation in type I but not in type II neurons, when they were depolarized from membrane potentials more negative than ‐60 mV. Bath application of NMDA (30–60 μM) elicited depolarization and triggered stable repetitive firing in all the NTS neurons but one. During the NMDA‐induced depolarization, hyperpolarization below ‐60 mV elicited, in some type I neurons, a rhythmic bursting pattern. The duration of the bursts (300–1000 ms) and their frequency (0.5–2 Hz) depended on the membrane potential. With hyperpolarizations below ‐75 mV, rhythmic bursting was converted into rhythmic single discharges, a pattern elicited directly in the other type I neurons. In all cases, rhythmic patterns were superimposed on cyclic depolarizations of the membrane potential characterized by an initial ramp‐shaped phase. In type II neurons, rhythmic bursting discharges, superimposed on rhythmic oscillations of the membrane potential, were also obtained upon hyperpolarization during the NMDA‐induced depolarization. In all type I neurons tested, NMDA‐induced cyclic ramp‐shaped depolarizations continued after addition of tetrodotoxin to the medium. Rhythmic bursting was not elicited by bath application of kainate (10–20 μM). Application of D‐2‐amino‐5‐phosphonovalerate (50 μM) blocked NMDA‐induced depolarizations without modifying those elicited by kainate, which were selectively depressed by 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (10 μM). Moreover, removal of Mg2+ from the medium suppressed NMDA‐induced cyclic depolarizations. Results demonstrate that both NMDA and non‐NMDA receptors are present in NTS neurons and that selective activation of NMDA receptors induced rhythmic bursting and/or rhythmic single discharges. Rhythmic patterns were not driven by synaptic mechanisms but originated from endogenous properties of NTS neurons activated by NMDA. Thus, NTS neurons can be considered as conditional pacemakers. According to the location of the neurons, the conditional properties shown in these in vitro experiments might be involved in vivo in the generation of rhythmic motor activities set up at the NTS level, such as swallowing.

Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics.

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.

Mixed GABA―glycine synapses delineate a specific topography in the nucleus tractus solitarii of adult rat

Using combined morphological and electrophysiological approaches, we have determined the composition of inhibitory synapses of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. Immunohistochemical experiments demonstrate that, in adult rat, GABA axon terminals are present throughout the NTS while mixed GABA–glycine axon terminals are strictly located to the lateral part of the NTS within subnuclei surrounding the tractus solitarius. Purely glycine axon terminals are rare in the lateral part of the NTS and hardly detected in its medial part. Electrophysiological experiments confirm the predominance of GABA inhibition throughout the NTS and demonstrate the existence of a dual inhibition involving the co‐release of GABA and glycine restricted to the lateral part of NTS. Since GABAA and glycine receptors are co‐expressed postsynaptically in virtually all the inhibitory axon terminals throughout the NTS, it suggests that the inhibition phenotype relies on the characteristics of the axon terminals. Our results also demonstrate that glycine is mostly associated with GABA within axon terminals and raise the possibility of a dynamic regulation of GABA/glycine release at the presynaptic level. Our data provide new information for understanding the mechanisms involved in the processing of visceral information by the central nervous system in adult animals.

Early expression of AMPA receptors and lack of NMDA receptors in developing rat climbing fibre synapses

Whether nascent glutamatergic synapses acquire their AMPA receptors constitutively or via a regulated pathway triggered by pre‐existing NMDA receptor activation is still an open issue. Here, we provide evidence that some glutamatergic synapses develop without expressing NMDA receptors. Using immunocytochemistry, we showed that synapses between developing rat climbing fibres and Purkinje cells expressed GluR2‐containing AMPA receptors as soon as they were formed (i.e. on embryonic day 19) but never carried detectable NMDA receptors. This was confirmed by electrophysiological recordings. Excitatory synaptic currents were recorded in Purkinje cells as early as P0. However, no NMDA receptor‐mediated component was found in either spontaneous or evoked synaptic responses. In addition, we ruled out a possible role of extrasynaptic NMDA receptors by showing that AMPA receptor clustering at nascent climbing fibre synapses was not modified by chronic in utero NMDA receptor blockade.

Perinatal development of inhibitory synapses in the nucleus tractus solitarii of the rat

The nucleus tractus solitarii (NTS) plays a key role in the central control of the autonomic nervous system. In adult rats, both GABA and glycine are used as inhibitory neurotransmitter in the NTS. Using a quantitative morphological approach, we have investigated the perinatal development of inhibitory synapses in the NTS. The density of both inhibitory axon terminals and synapses increased from embryonic day 20 until the end of the second postnatal week (postnatal day 14). Before birth, only GABAergic axon terminals developed and their number increased during the first postnatal week. Mixed GABA/glycine axon terminals appeared at birth and their number increased during the first postnatal week. This suggests the development of a mixed GABA/glycine inhibition in parallel to pure GABA inhibition. However, whereas GABAergic axon terminals were distributed throughout the NTS, mixed GABA/glycine axon terminals were strictly located in the lateral part of the NTS. Established at birth, this specific topography remained in the adult rat. From birth, GABAA receptors, glycine receptors and gephyrin were clustered in inhibitory synapses throughout the NTS, revealing a neurotransmitter–receptor mismatch within the medial part of the NTS. Together these results suggest that NTS inhibitory networks develop and mature until postnatal day 14. Developmental changes in NTS synaptic inhibition may play an important role in shaping neural network activity during a time of maturation of autonomic functions. The first two postnatal weeks could represent a critical period where the impact of the environment influences the physiological phenotypes of adult rats.

Synaptic localization of the glutamate receptor subunit GluR2 in the rat nucleus tractus solitarii

The GluR2 subunit controls several key features of the alpha amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)‐type glutamate receptor including calcium permeability, rectification and gating. In the present study, electrophysiological recordings and immunocytochemistry were used to document the synaptic localization of GluR2 in the rat nucleus tractus solitarii (NTS). Synaptic responses recorded in NTS neurons exhibited linear current–voltage relationships suggestive of GluR2‐containing AMPA receptor responses. Furthermore, after antigen retrieval GluR2 immunolabelling in the NTS mainly consisted of small puncta. Double‐labelling experiments showed that these GluR2 puncta were apposed to glutamatergic synaptic terminals identified by type II vesicular glutamate transporter immunoreactivity. These results indicate that NTS glutamatergic synapses are endowed with AMPA receptors which contain the GluR2 subunit and are therefore likely to be both calcium‐impermeable and slowly desensitizing.

Developmental Study of N-Methyl-d-Aspartate-induced Firing Activity and Whole-cell Currents in Nucleus Tractus Solitarii Neurons

Whole‐cell recordings of rat nucleus tractus solitarii (NTS) neurons were performed on a slice preparation. We investigated possible postnatal changes in firing activities and currents induced by N‐methyl‐d‐aspartate (NMDA) application. A total of 42 neurons were selected and fell into the following age groups: 0‐5 days (n= 15), 10‐15 days (n= 9) and 30‐60 days (adult, n= 18). During this period, input resistance and spike duration decreased by‐˜40%. At all ages, bath application of NMDA elicited a bursting firing activity when the membrane potential was held between ‐60 and ‐75 mV. However, in the youngest cells the rhythmic bursting activity was irregular and was characterized by a progressive firing inactivation during a burst. In a tetrodotoxin‐containing saline, NMDA‐induced oscillations of membrane potential were retained in all age groups. The membrane current‐voltage relationship of the NMDA‐induced inward current (INMDA) was characterized by a region of negative slope conductance which was similar in all age groups. Thus the voltage‐dependent block of INMDA is present in NTS neurons from birth, allowing NTS neurons to display membrane potential oscillations. However, postnatal maturation of repolarizing conductances, as suggested by changes in spike characteristics, could render the oscillatory activity more stable than at birth.