Frank P. Elsen

Respiratory rhythm generation in mammals: synaptic and membrane properties

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.

Emergent epileptiform activity in neural networks with weak excitatory synapses

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.

Prostaglandin E2-Induced Synaptic Plasticity in Neocortical Networks of Organotypic Slice Cultures

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.

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons

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‐Bötzinger 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 μm) 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 μm) decreased the HVA Ca2+ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca2+ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABAB receptor antagonist CGP 55845A (50 n m). 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.

When Norepinephrine Becomes a Driver of Breathing Irregularities: How Intermittent Hypoxia Fundamentally Alters the Modulatory Response of the Respiratory Network

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 both in vitro and in vivo by 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 output in vivo and 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.

Carbenoxolone induced depression of rhythmogenesis in the pre-Bötzinger Complex

BackgroundCarbenoxolone (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).ResultsWhereas 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.ConclusionThe 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.

Simulation of neocortical epileptiform activity using parallel computing

Abstract 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 differentially modulates the central control of eupnoea, sighs and gasping in mice

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.

Rhythmic intrinsic bursting neurons in human neocortex obtained from pediatric patients with epilepsy

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 (INaP) 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 INaP plays a critical role in intrinsic oscillatory activity observed in rIB neurons in the human neocortex. It is hypothesized that aberrant changes in INaP expression and/or function may ultimately contribute to neurological diseases that are linked to abnormal network activity, such as epilepsy.

Interaction between cellular voltage-sensitive conductance and network parameters in a model of neocortex can generate epileptiform bursting

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.