Membrane Potential Trajectories Research Articles

Impact of Unitary Synaptic Inhibition on Spike Timing in Ventral Tegmental Area Dopamine Neurons.

Midbrain dopamine neurons receive convergent synaptic input from multiple brain areas, which perturbs rhythmic pacemaking to produce the complex firing patterns observed in vivo. This study investigated the impact of single and multiple inhibitory inputs on ventral tegmental area (VTA) dopamine neuron firing in mice of both sexes using novel experimental measurements and modeling. We first measured unitary inhibitory postsynaptic currents produced by single axons using both minimal electrical stimulation and minimal optical stimulation of rostromedial tegmental nucleus and ventral pallidum afferents. We next determined the phase resetting curve, the reversal potential for GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs), and the average interspike membrane potential trajectory during pacemaking. We combined these data in a phase oscillator model of a VTA dopamine neuron, simulating the effects of unitary inhibitory postsynaptic conductances (uIPSGs) on spike timing and rate. The effect of a uIPSG on spike timing was predicted to vary according to its timing within the interspike interval or phase. Simulations were performed to predict the pause duration resulting from the synchronous arrival of multiple uIPSGs and the changes in firing rate and regularity produced by asynchronous uIPSGs. The model data suggest that asynchronous inhibition is more effective than synchronous inhibition, because it tends to hold the neuron at membrane potentials well positive to the IPSC reversal potential. Our results indicate that small fluctuations in the inhibitory synaptic input arriving from the many afferents to each dopamine neuron are sufficient to produce highly variable firing patterns, including pauses that have been implicated in reinforcement.

Persistent Interruption in Parvalbumin-Positive Inhibitory Interneurons: Biophysical and Mathematical Mechanisms.

Persistent activity in excitatory pyramidal cells (PYRs) is a putative mechanism for maintaining memory traces during working memory. We have recently demonstrated persistent interruption of firing in fast-spiking parvalbumin-expressing interneurons (PV-INs), a phenomenon that could serve as a substrate for persistent activity in PYRs through disinhibition lasting hundreds of milliseconds. Here, we find that hippocampal CA1 PV-INs exhibit type 2 excitability, like striatal and neocortical PV-INs. Modeling and mathematical analysis showed that the slowly inactivating potassium current KV1 contributes to type 2 excitability, enables the multiple firing regimes observed experimentally in PV-INs, and provides a mechanism for robust persistent interruption of firing. Using a fast/slow separation of times scales approach with the KV1 inactivation variable as a bifurcation parameter shows that the initial inhibitory stimulus stops repetitive firing by moving the membrane potential trajectory onto a coexisting stable fixed point corresponding to a nonspiking quiescent state. As KV1 inactivation decays, the trajectory follows the branch of stable fixed points until it crosses a subcritical Hopf bifurcation (HB) and then spirals out into repetitive firing. In a model describing entorhinal cortical PV-INs without KV1, interruption of firing could be achieved by taking advantage of the bistability inherent in type 2 excitability based on a subcritical HB, but the interruption was not robust to noise. Persistent interruption of firing is therefore broadly applicable to PV-INs in different brain regions but is only made robust to noise in the presence of a slow variable, KV1 inactivation.

Dendritic A-Current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic Cultures from Newborn Mice.

The brainstem preBötzinger complex (preBötC) generates the inspiratory rhythm for breathing. The onset of neural activity that precipitates the inspiratory phase of the respiratory cycle may depend on the activity of type-1 preBötC neurons, which exhibit a transient outward K+ current, IA Inspiratory rhythm generation can be studied ex vivo because the preBötC remains rhythmically active in vitro, both in acute brainstem slices and organotypic cultures. Advantageous optical conditions in organotypic slice cultures from newborn mice of either sex allowed us to investigate how IA impacts Ca2+ transients occurring in the dendrites of rhythmically active type-1 preBötC neurons. The amplitude of dendritic Ca2+ transients evoked via voltage increases originating from the soma significantly increased after an IA antagonist, 4-aminopyridine (4-AP), was applied to the perfusion bath or to local dendritic regions. Similarly, glutamate-evoked postsynaptic depolarizations recorded at the soma increased in amplitude when 4-AP was coapplied with glutamate at distal dendritic locations. We conclude that IA is expressed on type-1 preBötC neuron dendrites. We propose that IA filters synaptic input, shunting sparse excitation, while enabling temporally summated events to pass more readily as a result of IA inactivation. Dendritic IA in rhythmically active preBötC neurons could thus ensure that inspiratory motor activity does not occur until excitatory synaptic drive is synchronized and well coordinated among cellular constituents of the preBötC during inspiratory rhythmogenesis. The biophysical properties of dendritic IA might thus promote robustness and regularity of breathing rhythms.SIGNIFICANCE STATEMENT Brainstem neurons in the preBötC generate the oscillatory activity that underlies breathing. PreBötC neurons express voltage-dependent currents that can influence inspiratory activity, among which is a transient potassium current (IA) previously identified in a rhythmogenic excitatory subset of type-1 preBötC neurons. We sought to determine whether IA is expressed in the dendrites of preBötC. We found that dendrites of type-1 preBötC neurons indeed express IA, which may aid in shunting sparse non-summating synaptic inputs, while enabling strong summating excitatory inputs to readily pass and thus influence somatic membrane potential trajectory. The subcellular distribution of IA in rhythmically active neurons of the preBötC may thus be critical for producing well coordinated ensemble activity during inspiratory burst formation.

Adaptation in the visual cortex: influence of membrane trajectory and neuronal firing pattern on slow afterpotentials.

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p<0.05). Spike frequency adaptation had a faster time course and was larger during plateau (square pulse) than during intermittent (sinusoidal) depolarizations. Similar results were obtained in 17 neurons intracellularly recorded from the visual cortex in vivo. The differences in the afterpotentials evoked with both protocols were abolished by removing calcium from the extracellular medium or by application of the L-type calcium channel blocker nifedipine, suggesting that the activation of a calcium-dependent current is at the base of this afterpotential difference. These findings suggest that not only the spikes, but the membrane potential values and firing patterns evoked by a particular stimulation protocol determine the responses to any subsequent incoming input in a time window that spans for tens of seconds to even minutes.

Analysis of excitatory and inhibitory interactions at high temporal resolution in core circuits of the respiratory CPG

The pre-Botzinger complex (pre-BotC) is the essential core component of the brainstem respiratory central pattern generator (rCPG) in mammals. Phasic excitatory and inhibitory synaptic inputs during the respiratory cycle are thought to dynamically shape membrane potential trajectories and spiking patterns of pre-BotC neurons. These synaptic inputs reflect the functionally interacting neuron populations involved in rhythm and inspiratory-expiratory pattern generation. The dynamic patterns of these synaptic inputs have not been systematically studied and characterized experimentally. Accurate temporal reconstruction of the synaptic input patterns is important for testing current models of the rCPG that incorporate specific excitatory and inhibitory circuit mechanisms for rhythm and pattern generation. A recent computational model of the rCPG generating a normal “three-phase” respiratory pattern [1] specified patterns of synaptic excitation and inhibition in the preBotC during the respiratory cycle required to produce the behaviorally distinct phases of inspiration (I) and two phases of expiration (E1, or post-inspiration, and E2). This model has not been rigorously tested by experimental data on dynamic changes in phasic postsynaptic excitatory and inhibitory conductances in different populations of pre-BotC inspiratory and expiratory neurons. We developed analytical techniques that allow quantitative reconstruction of the patterns of synaptic conductances at high temporal resolution from intracellular recordings of membrane potential trajectories. Our approach extends previously proposed methods for extracting dynamic patterns of synaptic input conductances [2,3] and provides nearly continuous readouts of inhibitory and excitatory synaptic conductances in electrophysiologically different cell types throughout the respiratory cycle. The membrane potential trajectories of pre-BotC respiratory neurons were obtained by sharp microelectrode current-clamp recording within in situ perfused brainstem-spinal cord preparations of mature rats, which generate a three-phase respiratory pattern and provide mechanically stable conditions for intracellular recordings in functionally intact brainstem circuits. We distinguished different types of inspiratory and expiratory pre-BotC neurons and analyzed the dynamical changes of excitatory (Ge )a nd inhibitory (Gi) conductances. Inspiratory neurons exhibited strong excitatory inputs (large dynamic increases in Ge) during their spiking phase followed by a wave(s) of inhibitory inputs during the expiratory period with large increases in Gi and temporal patterns consistent with two populations of inhibitory neurons providing inputs that coordinate inspiratory to expiratory and expiratory to inspiratory phase transitions. Expiratory neurons exhibited strong inhibition during the inspiratory phase, consistent with the rhythmic alternation of inspiration and expiration, and changes in Ge indicating reciprocal interactions between two major populations of inhibitory neurons coordinating the generation of two (E1 and E2) phases of expiration. The reconstructed patterns of Ge and Gi are generally consistent with previously proposed circuit architecture [1] and also suggest its extension. The techniques that we have developed for high temporal resolution of postsynaptic conductance changes are likely to have broad application for analysis of synaptic interactions in circuit components whenever intracellular recordings of membrane potentials can be

Effective neuronal refractoriness dominates the statistics of superimposed spike trains

The pooled spike trains of populations of neurons are typically modeled as Poisson processes [2]. It is known, though, that the superposition of point processes is a Poisson process if and only if all components are Poisson processes [3]. However, neocortical neurons spike more regularly [1]. Partly this is because they often have a refractory period, but also because the membrane potential is hyperpolarized after each spike, as illustrated in Figure ​Figure1A.1A. Here we analyze neuronal spike trains recorded intracellularly in vivo from rat somatosensory cortex. We match them with a Poisson process with dead-time [4], which is the simplest model of neuronal activity that incorporates refractory effects. The dead-time here models the effective refractoriness of the neuron, which can be larger than the refractory period due to channel kinetics alone. From the spike train recordings we construct independent superpositions (see Figure ​Figure1B)1B) and compare their statistics to our analytical results for the model processes. We find that the effective refractoriness of the neurons dominates the second-order statistics of the superposition spike trains. We uncover profound statistical differences as compared to Poisson processes, which considerably affect the dynamics of the membrane potential of neurons that receive such superpositions, as we further show in numerical simulations (see also [5]). Figure 1 A: Membrane potential trajectories of a simulated neocortical neuron. After each spike, the potential has to charge up until spikes can be initiated by input fluctuations, leading to an effective refractoriness. Green line shows the mean subthreshold ...

Spiking behavior and membrane potential trajectories of pre‐BötC and hypoglossal neurons recorded from the rat in situ

Cellular and synaptic mechanisms operating in the brainstem respiratory network are not completely understood. Embedded in the respiratory network, the pre-Botzinger complex (pre-BötC) receives convergent inputs from numerous neuron populations. Hypoglossal motoneurons (XII MNs) are a major cranial motor output of the respiratory network and their activation during breathing, including by pre-BötC-driven circuits, is essential for maintaining upper airway patency. We applied sharp microelectrode intracellular recording techniques to characterize pre-BötC neuron and XII MN respiratory spiking patterns and input synaptic drives within in situ perfused brainstem-spinal cord preparations of mature rats, which allow analysis of patterning mechanisms during inspiratory and expiratory phases in an intact brainstem. A population of pre-BötC inspiratory neurons showed post-inspiratory hyperpolarization followed by ramping membrane potential and excitatory synaptic drive with characteristic pre-inspiratory spiking activity. In the XII motor nucleus we distinguished three groups of respiratory MNs by their spiking profile (including pre-inspiratory spiking) and membrane potential time course during the respiratory cycle. Our data suggest multiple pattern formation mechanisms and sources of synaptic drive shaping membrane potential trajectories of pre-BötC neurons and XII MNs in situ.

Estimates of EPSP amplitude based on changes in motoneuron discharge rate and probability

When motor units are discharging tonically, transient excitatory synaptic inputs produce an increase in the probability of spike occurrence and also increase the instantaneous discharge rate. Several researchers have proposed that these induced changes in discharge rate and probability can be used to estimate the amplitude of the underlying excitatory post-synaptic potential (EPSP). We tested two different methods of estimating EPSP amplitude by comparing the amplitude of simulated EPSPs with their effects on the discharge of rat hypoglossal motoneurons recorded in an in vitro brainstem slice preparation. The first estimation method (simplified-trajectory method) is based on the assumptions that the membrane potential trajectory between spikes can be approximated by a 10mV post-spike hyperpolarization followed by a linear rise to the next spike and that EPSPs sum linearly with this trajectory. We hypothesized that this estimation method would not be accurate due to interspike variations in membrane conductance and firing threshold that are not included in the model and that an alternative method based on estimating the effective distance to threshold would provide more accurate estimates of EPSP amplitude. This second method (distance-to-threshold method) uses interspike interval statistics to estimate the effective distance to threshold throughout the interspike interval and incorporates this distance-to-threshold trajectory into a threshold-crossing model. We found that the first method systematically overestimated the amplitude of small (<5mV) EPSPs and underestimated the amplitude of large (>5mV EPSPs). For large EPSPs, the degree of underestimation increased with increasing background discharge rate. Estimates based on the second method were more accurate for small EPSPs than those based on the first model, but estimation errors were still large for large EPSPs. These errors were likely due to two factors: (1) the distance to threshold can only be directly estimated over a limited portion of the interspike interval and (2) the distance to threshold can be affected by the EPSP itself. Both methods provide the most accurate EPSP estimates for EPSP amplitudes less than 5mV and moderate background discharge rates (~15imp/s).

Synapses with short-term plasticity are optimal estimators of presynaptic membrane potentials

The trajectory of the somatic membrane potential of a cortical neuron exactly reflects the computations performed on its afferent inputs. However, the spikes of such a neuron are a very low-dimensional and discrete projection of this continually evolving signal. We explored the possibility that the neuron’s efferent synapses perform the critical computational step of estimating the membrane potential trajectory from the spikes. We found that short-term changes in synaptic efficacy can be interpreted as implementing an optimal estimator of this trajectory. Short-term depression arose when presynaptic spiking was sufficiently intense as to reduce the uncertainty associated with the estimate; short-term facilitation reflected structural features of the statistics of the presynaptic neuron such as up and down states. Our analysis provides a unifying account of a powerful, but puzzling, form of plasticity.

A diverse pattern of the spike threshold changes in feline gastrocnemius–soleus motoneurons during stretch reflex activation

The aim of the study was to define the spike threshold changes in motoneurons during naturally evoked activation. Spike activity was evoked in the gastrocnemius-soleus motoneurons of decerebrate cats by controlled stretches of the homonymous muscles. The spike thresholds were defined by using the first derivative of the membrane potential and statistical boundaries of its change. The evoked firing was analyzed in terms of the spike thresholds and the membrane potential trajectories between spikes, which were dependent on the intensity of the firing. The main inference of the study is the absence of a single-valued dependence between the spike rates and thresholds; the last ones can both increase and decrease with a rise in the firing rate; moreover, opposite directions in the threshold changes are often observed at various phases of the stretch-evoked firing. The dependency of the spike thresholds on firing rates was checked in 42 of the total set of 57 motoneurons. In a group of cells (n = 27) showing statistically significant correlation (P < 0.05), this parameter was negative in 18 and positive in 9 motoneurons. The obtained results indicate that the threshold-crossing models with a single-valued dependency of the spike thresholds on the firing rates are not completely suitable to analyze the spike generation processes in the motor unit records.

Swallow-related inhibition in laryngeal motoneurons

Inhibitory postsynaptic potentials (IPSPs) of laryngeal motoneurons (LMs) are essential for narrowing the glottis at just the right time during swallowing, which prevents aspiration. To examine the property of IPSPs of LMs during swallowing, we monitored the effects of intracellular application of chloride ion and extracellular application of inhibitory neurotransmitter antagonists on the membrane potential trajectories of LMs during fictive swallowing in decerebrate, paralyzed cat. Adductor LMs hyperpolarized briefly at the beginning of the pharyngeal stage of swallowing (PS) and then depolarized explosively during the remaining part of the PS. Abductor LMs exhibited various patterns during swallowing; hyperpolarization during the PS followed by depolarization at the offset of the PS, slight depolarization, or plateau potentials. Chloride-dependent IPSPs were revealed during the initial part of PS in adductor LMs and during the whole PS in abductor LMs. The swallow-related IPSPs were depressed by iontophoretic extracellular application of bicuculline in both adductor and abductor LMs, but they were not modified by strychnine application. It is concluded that the swallow-related inhibition of both adductor and abductor LMs is chloride-dependent IPSPs mediated through GABA A receptors, not through glycine receptors.

How Gibbs distributions may naturally arise from synaptic adaptation mechanisms

It is assumed that complex perceptual or sensori-motor tasks are the result of neural network dynamics and are expressed by spike trains containing the neural code. Hence, in this context two main questions are (i) How to characterize the statistical properties of sequences the spikes trains produced by neuronal networks and (ii) What are the effects of synaptic plasticity on these statistics? Using methods from dynamical systems theory and statistical physics, we introduce a framework which applies for very general forms of spike train properties allowing to characterize miscellaneous forms of neural code (rank coding, synchronizations, correlations of different orders, etc.). In this framework, spike trains are associated with a coding of membrane potential trajectories constituting a symbolic coding in important modeling examples. On this basis, we use the thermodynamic formalism from ergodic theory to show how Gibbs distributions are natural probability measures to describe the statistics of spike trains, given the data of known empirical averages. Finally, we show that Gibbs distribution naturally arise when considering slow synaptic plasticity rules, where the only requirement is that characteristic time for synapse adaptation must be quite a bit longer that the characteristic time for neurons dynamics. We include some simulations results applying this framework on recurrent neural network with discrete time current based dynamics under the action of an STDP rule (see Figures 1 and 2). Numerical results are in accord with theory establishing that the topological pressure is a variation quantity with a minimum given by a Gibbs distribution (Figure 3).

How Gibbs Distributions May Naturally Arise from Synaptic Adaptation Mechanisms. A Model-Based Argumentation

This paper addresses two questions in the context of neuronal networks dynamics, using methods from dynamical systems theory and statistical physics: (i) How to characterize the statistical properties of sequences of action potentials ("spike trains") produced by neuronal networks ? and; (ii) what are the effects of synaptic plasticity on these statistics ? We introduce a framework in which spike trains are associated to a coding of membrane potential trajectories, and actually, constitute a symbolic coding in important explicit examples (the so-called gIF models). On this basis, we use the thermodynamic formalism from ergodic theory to show how Gibbs distributions are natural probability measures to describe the statistics of spike trains, given the empirical averages of prescribed quantities. As a second result, we show that Gibbs distributions naturally arise when considering "slow" synaptic plasticity rules where the characteristic time for synapse adaptation is quite longer than the characteristic time for neurons dynamics.

On dynamics of integrate-and-fire neural networks with conductance based synapses.

We present a mathematical analysis of networks with integrate-and-fire (IF) neurons with conductance based synapses. Taking into account the realistic fact that the spike time is only known within some finite precision, we propose a model where spikes are effective at times multiple of a characteristic time scale δ, where δ can be arbitrary small (in particular, well beyond the numerical precision). We make a complete mathematical characterization of the model-dynamics and obtain the following results. The asymptotic dynamics is composed by finitely many stable periodic orbits, whose number and period can be arbitrary large and can diverge in a region of the synaptic weights space, traditionally called the “edge of chaos”, a notion mathematically well defined in the present paper. Furthermore, except at the edge of chaos, there is a one-to-one correspondence between the membrane potential trajectories and the raster plot. This shows that the neural code is entirely “in the spikes” in this case. As a key tool, we introduce an order parameter, easy to compute numerically, and closely related to a natural notion of entropy, providing a relevant characterization of the computational capabilities of the network. This allows us to compare the computational capabilities of leaky and IF models and conductance based models. The present study considers networks with constant input, and without time-dependent plasticity, but the framework has been designed for both extensions.

Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats

There are at least two respiration-related rhythm generators in the medulla: the pre-Bötzinger complex, which produces inspiratory (Insp) neuron bursts, and the parafacial respiratory group (pFRG), which produces predominantly preinspiratory (Pre-I) neuron bursts. The pFRG Pre-I neuron activity has not been correlated with motor neuron activity in slice or block preparations of rostral medulla. In this study, we attempted to detect pFRG Pre-I activity as motor output in the rostral medulla. We recorded respiratory activity of the facial nerve in the brain stem-spinal cord preparation of 0- to 2-day-old rats. Facial nerve activity consisted of preinspiratory, Insp, and postinspiratory activity. Pre- and postinspiratory activity corresponded well with membrane potential trajectories of Pre-I neurons in the rostral ventrolateral medulla. In response to perfusion of 1 microM DAMGO (a mu-opiate agonist), fourth cervical ventral root (C4) Insp activity was depressed and facial nerve activity continued to synchronize with Pre-I neuron bursts. After transverse sectioning between the levels of the pre-Bötzinger complex and the pFRG, C4 Insp activity recovered within 15 min, but facial nerve activity was inhibited. When DAMGO was applied, C4 Insp activity was inhibited, and rhythmic facial nerve activity recovered. Subsequent elevation of K+ concentration reinduced C4 activity, but facial nerve activity was inhibited. Whole cell recordings in the rostral block revealed the presence of putative Pre-I neurons, the activity of which was synchronized with facial nerve activity. These results show that the rostral medulla, not including the pre-Bötzinger complex, produces Pre-I-like rhythmic activity that can be monitored as facial nerve motor output in newborn rat in vitro preparations.

Synaptic origin of the respiratory-modulated activity of laryngeal motoneurons

To determine the synaptic source of the respiratory-related activity of laryngeal motoneurons, spike-triggered averaging of the membrane potentials of laryngeal motoneurons was conducted using spikes of respiratory neurons located between the Bötzinger complex and the rostral ventral respiratory group as triggers in decerebrate, paralyzed cats. We identified one excitatory and two inhibitory sources for inspiratory laryngeal motoneurons, and two inhibitory sources for expiratory laryngeal motoneurons. In inspiratory laryngeal motoneurons, monosynaptic excitatory postsynaptic potentials were evoked by spikes of inspiratory neurons with augmenting firing patterns, and monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked by spikes of expiratory neurons with decrementing firing patterns and by spikes of inspiratory neurons with decrementing firing patterns. In expiratory laryngeal motoneurons, monosynaptic IPSPs were evoked by spikes of inspiratory neurons with decrementing firing patterns and by spikes of expiratory neurons with augmenting firing patterns. We conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of membrane potential of laryngeal motoneurons.

Ryanodine receptor/Ca 2+ release mechanisms in rhythmically active respiratory neurons of cats in vivo

The cytosolic Ca 2+ released from internal stores is important for distinctive cell functions. To assess the role of ryanodine/Ca 2+ releasing mechanisms in the rhythmic activity of respiratory neurons, effects of intracellular injection of ryanodine on the membrane potential trajectory of postinspiratory and augmenting inspiratory neurons were investigated in unanesthetized, decerebrate, paralyzed and artificially ventilated cats. Ryanodine injection hyperpolarized the membrane and decreased input resistance throughout the respiratory cycle in both types of respiratory neurons. Specifically, membrane repolarization during postinspiration was accelerated in postinspiratory neurons, and the large hyperpolarization at the onset of postinspiration was increased in augmenting inspiratory neurons. Spike-afterhyperpolarization consisting of a fast, early component and slow, late component increased in size after ryanodine, resulting in prolongation of inter-spike intervals and decrease of burst discharge. Intracellular injection of caffeine produced similar effects on these respiratory neurons, and Ruthenium Red, an antagonist of ryanodine receptors, had opposite effects. Immunoreactivity for ryanodine receptors was detected in all respiratory neurons labeled intracellularly with neurobiotin. These results demonstrate that ryanodine-sensitive Ca 2+ stores modulate the periodic membrane potential fluctuations and spike activity in respiratory neurons.

GABAergic and glycinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin-1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre-inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride-mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold-silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)- or glycine transporter 2 (GlyT2)-immunoreactive (ir) boutons and NK1R-ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD- and GlyT2-ir boutons were in close apposition to NK1R-ir somas and dendrites in the preBötC. Under the electron microscope, GAD- and GlyT2-ir terminals were in close apposition to NK1R-ir somas and dendrites. Symmetric synapses were identified between GAD- or GlyT2-ir terminals and NK1R-ir neurons. A total of 51.6% GAD-ir and 38.2% GlyT2-ir terminals were found to contact or make synapses with NK1R-ir profiles, respectively. GAD- and GlyT2-ir terminals synapsed not only upon NK1R-ir neurons but also upon NK1R immuno-negative neurons. NK1R-ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R-ir neurons in the preBötC, consistent with the suggestion that chloride-mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.

Respiratory pre-motor control of hypoglossal motoneurons in the rat

The goal of this study was to determine the origin and transmission pathway of respiratory drive to hypoglossal motoneurons. First we recorded intracellularly from 28 antidromically activated inspiratory hypoglossal motoneurons (resting membrane potential, −50±3 mV), and found that injection of chloride ions had no discernible effect on the shape of their membrane potential trajectories. We concluded that the membrane potential trajectories of these hypoglossal motoneurons were determined primarily by inspiratory excitation. To determine the origin of this excitation we cross-correlated the extracellular discharge of medullary inspiratory neurons, including those in the hypoglossal motor nucleus, with the hypoglossal nerve discharge. We found 27 inspiratory neurons within the hypoglossal motor nucleus that were not antidromically activated from the ipsilateral hypoglossal nerve; their cross-correlograms featured either central peaks (1.7±0.2 ms) alone ( n=14; 39%), or central peaks (1.3±0.2 ms) followed by troughs (1.3±0.1 ms) at short latencies (1.1±0.4 ms) ( n=13; 36%), and suggest that these neurons are hypoglossal interneurons. We recorded from 238 inspiratory neurons throughout the rest of the medulla; the cross-correlograms of 19 neurons (8%), located mostly in the lateral tegmental field, displayed narrow half-amplitude peaks (1.0±0.1 ms) at short latencies (0.9±0.1 ms), which we interpreted as evidence for monosynaptic excitation of hypoglossal motoneurons. We conclude that the respiratory control of hypoglossal motoneurons originates from inspiratory premotor neurons scattered throughout the lateral tegmental field and interneurons within the hypoglossal motor nucleus.

The respiratory rhythm in mutant oscillator mice

Since glycinergic inhibition is important for respiratory rhythm generation in mature mammals, we tested the hypothesis that the loss of glycine receptors during postnatal development (P17–P23) of homozygous mutant oscillator mice (spd ot/spd ot) may result in serious impairment of respiratory rhythm. We measured breathing in a plethysmographic recording chamber on conscious oscillator mice and used an in situ perfused brainstem preparation to record phrenic nerve activity, as well as membrane properties of respiratory neurones. The deletion of glycinergic inhibition did not result in failure of respiratory rhythm: homozygous mutant oscillator mice continue to generate a disturbed respiratory rhythm until death. Postsynaptic activity and membrane potential trajectories of respiratory neurones revealed a persistence of GABAergic inhibition and changes in respiratory rhythm and pattern generation.