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Prostaglandin E2 (PGE2) augments distinct inspiratory motor patterns, generated within the preBotzinger complex (preBotC), in a dose-dependent way. The frequency of sighs and gasping are stimulated at low concentrations, while the frequency of eupnoea increases only at high concentrations. We used in vivo microinjections into the preBotC and in vitro isolated brainstem slice preparations to investigate the dose-dependent effects of PGE2 on the preBotC activity. Synaptic measurements in whole cell voltage clamp recordings of inspiratory neurons revealed no changes in inhibitory or excitatory synaptic transmission in response to PGE2 exposure. In current clamp recordings obtained from inspiratory neurons of the preBotC, we found an increase in the frequency and amplitude of bursting activity in neurons with intrinsic bursting properties after exposure to PGE2. Riluzole, a blocker of the persistent sodium current, abolished the effect of PGE2 on sigh activity, while flufenamic acid, a blocker of the calcium-activated non-selective cation conductance, abolished the effect on eupnoeic activity caused by PGE2. Prostaglandins are important regulators of autonomic functions in the mammalian organism. Here we demonstrate in vivo that prostaglandin E2 (PGE2) can differentially increase the frequency of eupnoea (normal breathing) and sighs (augmented breaths) when injected into the preBotzinger complex (preBotC), a medullary area that is critical for breathing. Low concentrations of PGE2 (100-300 nm) increased the sigh frequency, while higher concentrations (1-2 μm) were required to increase the eupnoeic frequency. The concentration-dependent effects were similarly observed in the isolated preBotC. This in vitro preparation also revealed that riluzole, a blocker of the persistent sodium current (INap), abolished the modulatory effect on sighs, while flufenamic acid, an antagonist for the calcium-activated non-selective cation conductance (ICAN ) abolished the effect of PGE2 on fictive eupnoea at higher concentrations. At the cellular level PGE2 significantly increased the amplitude and frequency of intrinsic bursting in inspiratory neurons. By contrast PGE2 affected neither excitatory nor inhibitory synaptic transmission. We conclude that PGE2 differentially modulates sigh, gasping and eupnoeic activity by differentially increasing INap and ICAN currents in preBotC neurons.

Neocortical oscillations result from synchronized activity of a synaptically coupled network and can be strongly influenced by the intrinsic firing properties of individual neurons. As such, the intrinsic electroresponsive properties of individual neurons may have important implications for overall network function. Rhythmic intrinsic bursting (rIB) neurons are of particular interest, as they are poised to initiate and/or strongly influence network oscillations. Although neocortical rIB neurons have been recognized in multiple species, the current study is the first to identify and characterize rIB neurons in the human neocortex. Using whole-cell current-clamp recordings, rIB neurons (n = 12) are identified in human neocortical tissue resected from pediatric patients with intractable epilepsy. In contrast to human regular spiking neurons (n = 12), human rIB neurons exhibit rhythmic bursts of action potentials at frequencies of 0.1-4 Hz. These bursts persist after blockade of fast excitatory neurotransmission and voltage-gated calcium channels. However, bursting is eliminated by subsequent application of the persistent sodium current (I(NaP)) blocker, riluzole. In the presence of riluzole (either 10 or 20 μm), human rIB neurons no longer burst, but fire tonically like regular spiking neurons. These data demonstrate that I(NaP) plays a critical role in intrinsic oscillatory activity observed in rIB neurons in the human neocortex. It is hypothesized that aberrant changes in I(NaP) expression and/or function may ultimately contribute to neurological diseases that are linked to abnormal network activity, such as epilepsy.

Traumatic brain injury (TBI) is a major cause of epilepsy, yet the mechanisms underlying the progression from TBI to epilepsy are unknown. TBI induces the expression of COX-2 (cyclooxygenase-2) and increases levels of prostaglandin E2 (PGE2). Here, we demonstrate that acutely applied PGE2 (2 μm) decreases neocortical network activity by postsynaptically reducing excitatory synaptic transmission in acute and organotypic neocortical slices of mice. In contrast, long-term exposure to PGE2 (2 μm; 48 h) presynaptically increases excitatory synaptic transmission, leading to a hyperexcitable network state that is characterized by the generation of paroxysmal depolarization shifts (PDSs). PDSs were also evoked as a result of depriving organotypic slices of activity by treating them with tetrodotoxin (TTX, 1 μm; 48 h). This treatment predominantly increased postsynaptically excitatory synaptic transmission. The network and cellular effects of PGE2 and TTX treatments reversed within 1 week. Differences in the underlying mechanisms (presynaptic vs postsynaptic) as well as occlusion experiments in which slices were exposed to TTX plus PGE2 suggest that the two substances evoke distinct forms of homeostatic plasticity, both of which result in a hyperexcitable network state.PGE2 and TTX (alone or together with PGE2) also increased levels of apoptotic cell death in organotypic slices. Thus, we hypothesize that the increase in excitability and apoptosis may constitute the first steps in a cascade of events that eventually lead to epileptogenesis triggered by TBI.

Most types of electrographic epileptiform activity can be characterized by isolated or repetitive bursts in brain electrical activity. This observation is our motivation to determine mechanisms that underlie bursting behavior of neuronal networks. Here we show that the persistent sodium (NaP) current in mouse neocortical slices is associated with cellular bursting and our data suggest that these cells are capable of driving networks into a bursting state. This conclusion is supported by the following observations. 1) Both low concentrations of tetrodotoxin (TTX) and riluzole reduce and eventually stop network bursting while they simultaneously abolish intrinsic bursting properties and sensitivity levels to electrical stimulation in individual intrinsically bursting cells. 2) The sensitivity levels of regular spiking neurons are not significantly affected by riluzole or TTX at the termination of network bursting. 3) Propagation of cellular bursting in a neuronal network depended on excitatory connectivity and disappeared on bath application of CNQX (20 μM) + CPP (10 μM). 4) Voltage-clamp measurements show that riluzole (20 μM) and very low concentrations of TTX (50 nM) attenuate NaP currents in the neural membrane within a 1-min interval after bath application of the drug. 5) Recordings of synaptic activity demonstrate that riluzole at this concentration does not affect synaptic properties. 6) Simulations with a neocortical network model including different types of pyramidal cells, inhibitory interneurons, neurons with and without NaP currents, and recurrent excitation confirm the essence of our experimental observations that NaP conductance can be a critical factor sustaining slow population bursting.

Gamma-aminobutyric acid-type A receptors (GABA(A)-Rs) have been proposed as a target for many general anesthetics. We recently created knockin (KI) mice harboring a point mutation (serine 270 to histidine) in the GABA(A)-R alpha1 subunit. This mutation abolishes sensitivity of recombinant GABA(A)-Rs to isoflurane while maintaining normal sensitivity to halothane and increasing the potency of GABA. KI mice showed abnormalities in the EEG baseline, including occasional spike-wave activity and spindle-like bursts. When anesthetized with isoflurane, the KI mice but not the control mice revealed repetitive 4-5 Hz slow wave discharges in the cortical EEG. KI mice did not differ from controls in response to isoflurane or halothane in the standard tail clamp/withdrawal and loss of righting reflex assays. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from hippocampal interneurons and pyramidal cells in brain slices. mIPSCs in neurons from KI mice were of normal amplitude, but decayed more slowly than controls. Hippocampal mIPSCs in control mice were significantly prolonged by 0.4 and 0.9 MAC isoflurane, and by 0.5 MAC halothane. In KI mice, the effect of isoflurane on mIPSC decay was dramatically reduced, while halothane prolonged mIPSCs as for controls. We conclude that the kinetic and pharmacological properties of hippocampal GABA(A)-Rs in the KI mouse recapitulate many features of mutant alpha1beta2gamma2 GABA(A)-Rs observed in vitro. GABA(A)-Rs containing alpha1 subunits do not appear to contribute to the actions of isoflurane in the spinal cord, but both EEG and synaptic recordings provide evidence for effects of isoflurane on these GABA(A)-R isoforms in cortical structures.

The mammalian respiratory network reorganizes during early postnatal life. We characterized the postnatal developmental changes of calcium currents in neurons of the pre-Bötzinger complex (pBC), the presumed site for respiratory rhythm generation. The pBC contains not only respiratory rhythmic (R) but also nonrhythmic neurons (nR). Both types of neurons express low- and high-voltage-activated (LVA and HVA) calcium currents. This raises the interesting issue: do calcium currents of the two co-localized neuron types have similar developmental profiles? To address this issue, we used the whole cell patch-clamp technique to compare in transverse slices of mice LVA and HVA calcium current amplitudes of the two neuron populations (R and nR) during the first and second postnatal week (P0–P16). The amplitude of HVA currents did not significantly change in R pBC-neurons (P0–P16), but it significantly increased in nR pBC-neurons during P8–P16. The dehydropyridine (DHP)-sensitive current amplitudes did not significantly change during the early postnatal development, suggesting that the observed amplitude changes in nR pBC-neurons are caused by (DHP) insensitive calcium currents. The ratio between HVA calcium current amplitudes dramatically changed during early postnatal development: At P0–P3, current amplitudes were significantly larger in R pBC-neurons, whereas at P8–P16, current amplitudes were significantly larger in nR pBC-neurons. Our results suggest that calcium currents in pBC neurons are differentially altered during postnatal development and that R pBC-neurons have fully expressed calcium currents early during postnatal development. This may be critical for stable respiratory rhythm generation in the underlying rhythm generating network.

A scalable network model intended for study of neocortical epileptiform activity was built on the pGENESIS neural simulator. The model included superficial and deep pyramidal cells plus four types of inhibitory neurons. An electroencephalogram (EEG) simulator was attached to the model to validate model behavior and to determine the contributions of inhibitory and excitatory neuronal populations to the EEG signal. We examined effects of overall excitation and inhibition on activity patterns in the network, and found that the network-bursting patterns occur within a narrow range of the excitation–inhibition space. Further, we evaluated synchronization effects produced by gap junctions during synchronous and asynchronous states.

Prostaglandin E2 (PGE2) augments distinct inspiratory motor patterns, generated within the preBötzinger complex (preBötC), in a dose-dependent way. The frequency of sighs and gasping are stimulated at low concentrations, while the frequency of eupnoea increases only at high concentrations. We used in vivo microinjections into the preBötC and in vitro isolated brainstem slice preparations to investigate the dose-dependent effects of PGE2 on the preBötC activity. Synaptic measurements in whole cell voltage clamp recordings of inspiratory neurons revealed no changes in inhibitory or excitatory synaptic transmission in response to PGE2 exposure. In current clamp recordings obtained from inspiratory neurons of the preBötC, we found an increase in the frequency and amplitude of bursting activity in neurons with intrinsic bursting properties after exposure to PGE2. Riluzole, a blocker of the persistent sodium current, abolished the effect of PGE2 on sigh activity, while flufenamic acid, a blocker of the calcium-activated non-selective cation conductance, abolished the effect on eupnoeic activity caused by PGE2. Prostaglandins are important regulators of autonomic functions in the mammalian organism. Here we demonstrate in vivo that prostaglandin E2 (PGE2) can differentially increase the frequency of eupnoea (normal breathing) and sighs (augmented breaths) when injected into the preBötzinger complex (preBötC), a medullary area that is critical for breathing. Low concentrations of PGE2 (100-300 nm) increased the sigh frequency, while higher concentrations (1-2 μm) were required to increase the eupnoeic frequency. The concentration-dependent effects were similarly observed in the isolated preBötC. This in vitro preparation also revealed that riluzole, a blocker of the persistent sodium current (INap), abolished the modulatory effect on sighs, while flufenamic acid, an antagonist for the calcium-activated non-selective cation conductance (ICAN ) abolished the effect of PGE2 on fictive eupnoea at higher concentrations. At the cellular level PGE2 significantly increased the amplitude and frequency of intrinsic bursting in inspiratory neurons. By contrast PGE2 affected neither excitatory nor inhibitory synaptic transmission. We conclude that PGE2 differentially modulates sigh, gasping and eupnoeic activity by differentially increasing INap and ICAN currents in preBötC neurons.

Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation. Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these “functional” in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of −60 mV evoked in control neurons potassium currents with a peak current of 10.0 ± 2.5 nA and a large steady state current of 8.5 ± 2.6 nA, which was still activated from a holding potential of −40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 ± 2.4 nA; steady state: 3.6 ± 2.0 nA). This current was inactivated at a holding potential of −40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42°C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices.

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca2+-dependent K+ channels via Ca2+-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba2+ and the reversal of its underlying current was significantly shifted towards EK+, suggesting that it is due to the activation of K+ channels. (2) The delayed second conductance increase disappeared in a Ca2+-free saline buffered with BAPTA, indicating that it depended on Ca2+-influx. (3) Co2+, Cd2+ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca2+ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca2+ stores with the Ca2+-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca2+ influx that could lead indirectly to the activation of Ca2+-dependent K+ channels.

Neuronal networks are endogenously modulated by aminergic and peptidergic substances. These modulatory processes are critical for maintaining normal activity and adapting networks to changes in metabolic, behavioral, and environmental conditions. However, disturbances in neuromodulation have also been associated with pathologies. Using whole animals (in vivo) and functional brainstem slices (in vitro) from mice, we demonstrate that exposure to acute intermittent hypoxia (AIH) leads to fundamental changes in the neuromodulatory response of the respiratory network located within the preBötzinger complex (preBötC), an area critical for breathing. Norepinephrine, which normally regularizes respiratory activity, renders respiratory activity irregular after AIH. Respiratory irregularities are caused bothin vitroandin vivoby AIH, which increases synaptic inhibition within the preBötC when norepinephrine is endogenously or exogenously increased. These irregularities are prevented by blocking synaptic inhibition before AIH. However, regular breathing cannot be reestablished if synaptic inhibition is blocked after AIH. We conclude that subtle changes in synaptic transmission can have dramatic consequences at the network level as endogenously released neuromodulators that are normally adaptive become the drivers of irregularity. Moreover, irregularities in the preBötC result in irregularities in the motor outputin vivoand in incomplete transmission of inspiratory activity to the hypoglossus motor nucleus. Our finding has basic science implications for understanding network functions in general, and it may be clinically relevant for understanding pathological disturbances associated with hypoxic episodes such as those associated with myocardial infarcts, obstructive sleep apneas, apneas of prematurity, Rett syndrome, and sudden infant death syndrome.

Neocortical oscillations result from synchronized activity of a synaptically coupled network and can be strongly influenced by the intrinsic firing properties of individual neurons. As such, the intrinsic electroresponsive properties of individual neurons may have important implications for overall network function. Rhythmic intrinsic bursting (rIB) neurons are of particular interest, as they are poised to initiate and/or strongly influence network oscillations. Although neocortical rIB neurons have been recognized in multiple species, the current study is the first to identify and characterize rIB neurons in the human neocortex. Using whole-cell current-clamp recordings, rIB neurons (n = 12) are identified in human neocortical tissue resected from pediatric patients with intractable epilepsy. In contrast to human regular spiking neurons (n = 12), human rIB neurons exhibit rhythmic bursts of action potentials at frequencies of 0.1-4 Hz. These bursts persist after blockade of fast excitatory neurotransmission and voltage-gated calcium channels. However, bursting is eliminated by subsequent application of the persistent sodium current (I(NaP)) blocker, riluzole. In the presence of riluzole (either 10 or 20 μm), human rIB neurons no longer burst, but fire tonically like regular spiking neurons. These data demonstrate that I(NaP) plays a critical role in intrinsic oscillatory activity observed in rIB neurons in the human neocortex. It is hypothesized that aberrant changes in I(NaP) expression and/or function may ultimately contribute to neurological diseases that are linked to abnormal network activity, such as epilepsy.

Background Carbenoxolone (CBX), a gap junction uncoupler, alters the functioning of the pre-Bötzinger Complex (preBötC), a central pattern generating neuronal network important for the production of respiratory rhythm in mammals. Even when isolated in a 1/2 mm-thick slice of medulla oblongata from neonatal mouse the preBötC continues producing periodic bursts of action potentials, termed population bursts that are thought to be important in generating various patterns of inspiration, in vivo. Whether gap junction communication contributes to preBötC rhythmogenesis remains unresolved, largely because existing gap junction uncouplers exert numerous non-specific effects (e.g., inhibition of active transport, alteration of membrane conductances). Here, we determined whether CBX alters preBötC rhythmogenesis by altering membrane properties including input resistance (Rin), voltage-gated Na+ current (INa), and/or voltage-gated K+ current (IK), rather than by blocking gap junction communication. To do so we used a medullary slice preparation, network-level recordings, whole-cell voltage clamp, and glycyrrhizic acid (GZA; a substance used as a control for CBX, since it is similar in structure and does not block gap junctions). Results Whereas neither of the control treatments [artificial cerebrospinal fluid (aCSF) or GZA (50 μM)] noticeably affected preBötC rhythmogenesis, CBX (50 μM) decreased the frequency, area and amplitude of population bursts, eventually terminating population burst production after 45–60 min. Both CBX and GZA decreased neuronal Rin and induced an outward holding current. Although neither agent altered the steady state component of IK evoked by depolarizing voltage steps, CBX, but not GZA, increased peak INa. Conclusion The data presented herein are consistent with the notion that gap junction communication is important for preBötC rhythmogenesis. By comparing the effects of CBX and GZA on membrane properties our data a) demonstrate that depression of preBötC rhythmogenesis by CBX results from actions on another variable or other variables; and b) show that this comparative approach can be used to evaluate the potential contribution of other non-specific actions (e.g., Ca++ conductances or active transport) of CBX, or other uncouplers, in their alteration of preBötC rhythmogenesis, or the functioning of other networks.

Background Enhancement of the function of gamma-aminobutyric acid type A receptors containing the alpha1 subunit may underlie a portion of inhaled anesthetic action. To test this, the authors created gene knock-in mice harboring mutations that render the receptors insensitive to isoflurane while preserving sensitivity to halothane. Methods The authors recorded miniature inhibitory synaptic currents in hippocampal neurons from hippocampal slices from knock-in and wild-type mice. They also determined the minimum alveolar concentration (MAC), and the concentration at which 50% of animals lost their righting reflexes and which suppressed pavlovian fear conditioning to tone and context in both genotypes. Results Miniature inhibitory postsynaptic currents decayed more rapidly in interneurons and CA1 pyramidal cells from the knock-in mice compared with wild-type animals. Isoflurane (0.5-1 MAC) prolonged the decay phase of miniature inhibitory postsynaptic currents in neurons of the wild-type mice, but this effect was significantly reduced in neurons from knock-in mice. Halothane (1 MAC) slowed the decay of miniature inhibitory postsynaptic current in both genotypes. The homozygous knock-in mice were more resistant than wild-type controls to loss of righting reflexes induced by isoflurane and enflurane, but not to halothane. The MAC for isoflurane, desflurane, and halothane did not differ between knock-in and wild-type mice. The knock-in mice and wild-type mice did not differ in their sensitivity to isoflurane for fear conditioning. Conclusions gamma-Aminobutyric acid type A receptors containing the alpha1 subunit participate in the inhibition of the righting reflexes by isoflurane and enflurane. They are not, however, involved in the amnestic effect of isoflurane or immobilizing actions of inhaled agents.

Brain electrical activity recorded during an epileptic seizure is frequently associated with rhythmic discharges in cortical networks. Current opinion in clinical neurophysiology is that strongly coupled networks and cellular bursting are prerequisites for the generation of epileptiform activity. Contrary to expectations, we found that weakly coupled cortical networks can create synchronized cellular activity and seizure-like bursting. Evaluation of a range of synaptic parameters in a detailed computational model revealed that seizure-like activity occurs when the excitatory synapses are weakened. Guided by this observation, we confirmed experimentally that, in mouse neocortical slices, a pharmacological reduction of excitatory synaptic transmission elicited sudden onset of repetitive network bursting. Our finding provides powerful evidence that onset of seizures can be associated with a reduction in synaptic transmission. These results open a new avenue to explore network synchrony and may ultimately lead to a rational approach to treatment of network pathology in epilepsy.

We examined the effects of both intrinsic neuronal membrane properties and network parameters on oscillatory activity in a model of neocortex. A scalable network model with six different cell types was built with the pGENESIS neural simulator. The neocortical network consisted of two types of pyramidal cells and four types of inhibitory interneurons. All cell types contained both fast sodium and delayed rectifier potassium channels for generation of action potentials. A subset of the pyramidal neurons contained an additional slow inactivating (persistent) sodium current (NaP). The neurons with the NaP current showed spontaneous bursting activity in the absence of external stimulation. The model also included a routine to calculate a simulated electroencephalogram (EEG) trace from the population activity. This revealed emergent network behavior which ranged from desynchronized activity to different types of seizure-like bursting patterns. At settings with weaker excitatory network effects, the propensity to generate seizure-like behavior increased. Strong excitatory network connectivity destroyed oscillatory behavior, whereas weak connectivity enhanced the relative importance of the spontaneously bursting cells. Our findings are in contradiction with the general opinion that strong excitatory synaptic and/or insufficient inhibition effects are associated with seizure initiation, but are in agreement with previously reported behavior in neocortex.

GABAAreceptors (GABAA-Rs) are pentameric structures consisting of two α, two β, and one γ subunit. The α subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the α1 subunit to native GABAA-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and α1 subunit knock-out (α1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from α1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the α1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from α1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the α1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the α1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.

GABAB receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABAB receptor activation on voltage-activated Ca2+ currents were examined in rhythmically active neurons of the pre-Botzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca2+ currents were present from the first postnatal day (P1). The density of LVA Ca2+ currents increased during the first week, whilst the density of HVA Ca2+ currents increased after the first week. In the second postnatal week, the HVA Ca2+ currents were composed of L- (47 +/- 10%) and N-type (21 +/- 8%) currents plus a 'residual' current, whilst there were no N-type currents detectable in the first few days. The GABAB receptor agonist baclofen (30 microM) increased LVA Ca2+ currents (30 +/- 11%) at P1-P3, but it decreased the currents (35 +/- 11%) at P7-P15 without changing its time course. At all ages, baclofen (30 microM) decreased the HVA Ca2+ currents by approximately 54%. Threshold of baclofen effects on both LVA and HVA Ca2+ currents was 5 microM at P1-P3 and lower than 1 microM at P7-P15. The effect of baclofen was abolished in the presence of the GABAB receptor antagonist CGP 55845A (50 nM). We conclude that both LVA and HVA Ca2+ currents increased postnatally. The GABAB receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.

To obtain a quantitative characterization of voltage-activated calcium currents in respiratory neurons, we performed voltage-clamp recordings in the transverse brainstem slice of mice from neurons located within the ventral respiratory group. It is assumed that this medullary region contains the neuronal network responsible for generating the respiratory rhythm. This study represents one of the first attempts to analyze quantitatively the currents in respiratory neurons. The inward calcium currents of VRG neurons consisted of two components: a high voltage-activated (HVA) and a low voltage-activated (LVA) calcium current. The activation threshold of the HVA current was at −40 mV. It was fully activated (peak voltage) between −10 and 0 mV. The half-maximal activation (V50) was at −27.29 mV ± 1.15 (n= 24). The HVA current was inactivated completely at a holding potential of −35 mV and fully deinactivated at a holding potential of −65 mV (V50, −52.26 mV ± 0.27;n= 18). The threshold for the activation of the LVA current was at −65 mV. This current had its peak voltage between −50 and −40 mV (mean,V50= −59.15 mV ± 0.21;n= 15). The LVA current was inactivated completely at a holding potential of −65 mV and deinactivated fully at a holding potential of −95 mV (mean,V50= −82.40 mV ± 0.32;n= 38). These properties are consistent with other studies suggesting that the LVA current is a T-type current. The properties of these inward currents are discussed with respect to their role in generating Ca2+potentials that may contribute to the generation of the mammalian respiratory rhythm.

Respiratory rhythm generation depends on a complex interaction between synaptic and membrane properties of functionally defined neurons. To gain a better understanding of how inhibitory and excitatory synaptic inputs lead to the generation of the respiratory rhythm we analyzed the depolarization pattern of respiratory neurons that were recorded in the transverse slice preparation of mice (P8-22) and the in vivo adult cat. Using voltage-calmp recordings from respiratory neurons and specific antagonists for inhibitory synaptic transmission we demonstrate under in vitro conditions, that inspiratory (n = 7) and post-inspiratory neurons (n = 13) received concurrent glycinergic and glutamatergic synaptic input during inspiration. A similar conclusion was gained with chloride injections into in vivo respiratory neurons. The inhibitory input was essential not only for generating the characteristic depolarization pattern of respiratory neurons, but also for switching the respiratory rhythm between inspiration and post-inspiration. The generation of the depolarization pattern depends also on intrinsic membrane properties. Negative current injections reveal that excitatory synaptic input was amplified by intrinsic bursting properties in some inspiratory neurons (n = 4) recorded in vitro. Although such properties have not been described under in vivo conditions our findings suggest that with respect to inspiratory, post-inspiratory and late-inspiratory neurons, the principle network organization is similar under both in vitro and in vivo conditions.