Fernando Peña

Differential contribution of pacemaker properties to the generation of respiratory rhythms during normoxia and hypoxia

Pacemaker neurons have been described in most neural networks. However, whether such neurons are essential for generating an activity pattern in a given preparation remains mostly unknown. Here, we show that in the mammalian respiratory network two types of pacemaker neurons exist. Differential blockade of these neurons indicates that their relative contribution to respiratory rhythm generation changes during the transition from normoxia to hypoxia. During hypoxia, blockade of neurons with sodium-dependent bursting properties abolishes respiratory rhythm generation, while in normoxia respiratory rhythm generation only ceases upon pharmacological blockade of neurons with heterogeneous bursting properties. We propose that respiratory rhythm generation in normoxia depends on a heterogeneous population of pacemaker neurons, while during hypoxia the respiratory rhythm is driven by only one type of pacemaker.

Mecp2 Deficiency Disrupts Norepinephrine and Respiratory Systems in Mice

Rett syndrome is a severe X-linked neurological disorder in which most patients have mutations in the methyl-CpG binding protein 2 (MECP2) gene and suffer from bioaminergic deficiencies and life-threatening breathing disturbances. We used in vivo plethysmography, in vitro electrophysiology, neuropharmacology, immunohistochemistry, and biochemistry to characterize the consequences of the MECP2 mutation on breathing in wild-type (wt) and Mecp2-deficient (Mecp2-/y) mice. At birth, Mecp2-/y mice showed normal breathing and a normal number of medullary neurons that express tyrosine hydroxylase (TH neurons). At ∼1 month of age, most Mecp2-/y mice showed respiratory cycles of variable duration; meanwhile, their medulla contained a significantly reduced number of TH neurons and norepinephrine (NE) content, even in Mecp2-/y mice that showed a normal breathing pattern. Between 1 and 2 months of age, all unanesthetized Mecp2-/y mice showed breathing disturbances that worsened until fatal respiratory arrest at ∼2 months of age. During their last week of life, Mecp2-/y mice had a slow and erratic breathing pattern with a highly variable cycle period and frequent apneas. In addition, their medulla had a drastically reduced number of TH neurons, NE content, and serotonin (5-HT) content. In vitro experiments using transverse brainstem slices of mice between 2 and 3 weeks of age revealed that the rhythm produced by the isolated respiratory network was irregular in Mecp2-/y mice but could be stabilized with exogenous NE. We hypothesize that breathing disturbances in Mecp2-/y mice, and probably Rett patients, originate in part from a deficiency in noradrenergic and serotonergic modulation of the medullary respiratory network.

Pacemaker neurons and neuronal networks: an integrative view.

Rhythmically active neuronal networks give rise to rhythmic motor activities but also to seemingly non-rhythmic behaviors such as sleep, arousal, addiction, memory and cognition. Many of these networks contain pacemaker neurons. The ability of these neurons to generate bursts of activity intrinsically lies in voltage- and time-dependent ion fluxes resulting from a dynamic interplay among ion channels, second messenger pathways and intracellular Ca2+ concentrations, and is influenced by neuromodulators and synaptic inputs. This complex intrinsic and extrinsic modulation of pacemaker activity exerts a dynamic effect on network activity. The nonlinearity of bursting activity might enable pacemaker neurons to facilitate the onset of excitatory states or to synchronize neuronal ensembles--an interactive process that is intimately regulated by synaptic and modulatory processes.

Gasping Activity In Vitro: A Rhythm Dependent on 5-HT2A Receptors

Many rhythmic behaviors are continuously modulated by endogenous peptides and amines, but whether neuromodulation is critical to the expression of a rhythmic behavior often remains unknown, particularly in mammals. Here, we address this issue in the respiratory network that was isolated in spontaneously rhythmic medullary slice preparations from mice. Under control conditions, the respiratory network generates fictive eupneic activity. We hypothesized previously that this activity depends on two types of pacemaker neurons. The bursting properties of one pacemaker rely on the persistent sodium current (INa(p)) and are insensitive to blockade of calcium channels with cadmium (CI-pacemakers), whereas bursting mechanisms of a second pacemaker are sensitive to cadmium (CS-pacemakers) and the calcium-dependent nonspecific cation current blocker flufenamic acid. During hypoxia, fictive eupneic activity is supplanted by the neural correlate of gasping, which is proposed to depend only on CI-pacemakers. Because CI-pacemakers require endogenous activation of 5-HT2A receptors, we tested the hypothesis that 5-HT2A receptor activation is critical for gasping. Here, we demonstrate that fictive gasping and CI-pacemaker bursting were selectively eliminated by the 5-HT2A receptor antagonist piperidine or ketanserin. Neither 5-HT2A antagonist eliminated bursting by CS-pacemakers and ventral respiratory group (VRG) population activity. However, this VRG activity was very different from eupneic activity. In the presence of 5-HT2A antagonists, VRG activity was eliminated by flufenamic acid and could not be reliably restored by adding substance P. These data support the hypothesis that two types of pacemaker bursting mechanisms underlie fictive eupnea, whereas only one burst mechanism is critical for gasping.

Substance P-Mediated Modulation of Pacemaker Properties in the Mammalian Respiratory Network

Neuromodulators are integral parts of a neuronal network, and unraveling how these substances alter neuronal activity is critical for understanding how networks generate patterned activity and, ultimately, behavior. In this study, we examined the cellular mechanisms underlying the excitatory action of substance P (SP) on the respiratory network isolated in spontaneously active transverse slice preparation of mice. SP produced a slow depolarization in all recorded inspiratory pacemaker and non-pacemaker neurons. Ion exchange experiments and blockers for different ion channels suggest that the slow depolarization is caused by the activation of a low-threshold TTX-insensitive cationic current that carries mostly Na+. The SP-induced slow depolarization increased tonic discharge in non-pacemaker neurons and primarily enhanced the frequency of bursting in Cd2+-insensitive pacemaker neurons. In the Cd2+-sensitive pacemaker neuron, the burst frequency was not significantly affected, whereas burst duration and amplitude were more enhanced than in Cd2+-insensitive pacemaker neurons. In a subset of non-pacemaker neurons that produced NMDA-dependent subthreshold oscillations, SP caused the production of bursts of action potentials. We conclude that the degree of pacemaker activity in the respiratory network is not fixed but dynamically regulated by neuromodulators such as SP. This finding may have clinical implications for Rett syndrome in which SP levels along with other neuromodulators are decreased in the brainstem.

Differential modulation of neural network and pacemaker activity underlying eupnea and sigh-breathing activities

Many networks generate distinct rhythms with multiple frequency and amplitude characteristics. The respiratory network in the pre-Bötzinger complex (pre-Böt) generates both the low-frequency, large-amplitude sigh rhythm and a faster, smaller-amplitude eupneic rhythm. Could the same set of pacemakers generate both rhythms? Here we used an in vitro respiratory brainslice preparation. We describe a subset of synaptically isolated pacemakers that spontaneously generate two distinct bursting patterns. These two patterns resemble network activity including sigh-like bursts that occur at low frequencies and have large amplitudes and eupneic-like bursts with higher frequency and smaller amplitudes. Cholinergic neuromodulation altered the network and pacemaker bursting: fictive sigh frequency is increased dramatically, whereas fictive eupneic frequency is drastically lowered. The data suggest that timing and amplitude characteristics of fictive eupneic and sigh rhythms are set by the same set of pacemakers that are tuned by changes in the neuromodulatory state.

Hypoxia-induced changes in neuronal network properties

Because of their high energetic demand, neurons within the mammalian central nervous system are extremely sensitive to changes in partial pressure of oxygen. Faced with acute hypoxic conditions, an organism must follow a complex and highly dynamic emergency plan to secure survival. Behavioral functions that are not immediately essential for survival are turned off, and critical behaviors (such as breathing) undergo a biphasic response. An augmentation of breathing is initially adaptive, whereas prolonged hypoxic conditions are better served by an energy-saving mode. However, the hypoxic response of an organism depends on many additional factors. Environmental conditions, the animal’s age and health, and the pattern (continuous vs intermittent) and duration (chronic vs acute) of hypoxia greatly determine the specific course of a hypoxic response. Different forms of hypoxia can cause pathology or be used as therapy. Therefore, it is not surprising that the hypoxic response of an organism results from widespread and highly diverse reconfigurations of neuronal network functions in different brain areas that are accomplished by diverse hypoxic changes at all levels of the nervous system (i.e., molecular, cellular, synaptic, neuronal, network). Hypoxia-induced changes in synaptic transmission are generally depressive and result primarily from presynaptic mechanisms, whereas changes in intrinsic properties involve excitatory and inhibitory alterations involving the majority of K+, Na+, and Ca2+ channels. This article reviews the response of the nervous system to hypoxia, accounting for all levels of integration from the cellular to the network level, and postulates that a better understanding of the diversity of cellular events is only possible if cellular and network events are considered in a functional and organismal context.

Beta-amyloid protein (25-35) disrupts hippocampal network activity: role of Fyn-kinase.

Early cognitive deficit characteristic of early Alzheimers disease seems to be produced by the soluble forms of β‐amyloid protein. Such cognitive deficit correlates with neuronal network dysfunction that is reflected as alterations in the electroencephalogram of both Alzheimer patients and transgenic murine models of such disease. Correspondingly, recent studies have demonstrated that chronic exposure to βAP affects hippocampal oscillatory properties. However, it is still unclear if such neuronal network dysfunction results from a direct action of βAP on the hippocampal circuit or it is secondary to the chronic presence of the protein in the brain. Therefore, we aimed to explore the effect of acute exposure to βAP25–35 on hippocampal network activity both in vitro and in vivo, as well as on intrinsic and synaptic properties of hippocampal neurons. We found that βAP25–35, reversibly, affects spontaneous hippocampal population activity in vitro. Such effect is not produced by the inverse sequence βAP35–25 and is reproduced by the full‐length peptide βAP1–42. Correspondingly βAP25–35, but not the inverse sequence βAP35–25, reduces theta‐like activity recorded from the hippocampus in vivo. The βAP25–35‐induced disruption in hippocampal network activity correlates with a reduction in spontaneous neuronal activity and synaptic transmission, as well as with an inhibition in the subthreshold oscillations produced by pyramidal neurons in vitro. Finally, we studied the involvement of Fyn‐kinase on the βAP25–35‐induced disruption in hippocampal network activity in vitro. Interestingly, we found that such phenomenon is not observed in slices obtained from Fyn‐knockout mice. In conclusion, our data suggest that βAP acutely affects proper hippocampal function through a Fyn‐dependent mechanism. We propose that such alteration might be related to the cognitive impairment observed, at least, during the early phases of Alzheimers disease.

Effects of riluzole and flufenamic acid on eupnea and gasping of neonatal mice in vivo.

The pre-Bötzinger complex (PBC), part of the ventral respiratory group that is responsible for inspiratory rhythm generation, contains at least two types of pacemaker neurons. In vitro studies have shown that bursting properties of one type of pacemaker relies on a riluzole-sensitive persistent sodium current, whereas bursting of a second type is sensitive to flufenamic acid (FFA), a calcium-dependent nonspecific cationic current blocker. In vitro, under control conditions, the PBC generates fictive eupneic activity that depends on both riluzole-sensitive and FFA-sensitive pacemaker neurons. During hypoxia the PBC generates fictive gasping activity and only riluzole-sensitive pacemaker neurons appear to be necessary for this rhythm. We carried out pharmacological experiments to test the role of respiratory pacemaker neurons in vivo by performing plethysmographic recordings on neonate mice. As reported in vitro, eupnea activity in vivo is abolished only if both FFA and riluzole are coadministered intracisternally, but not when either of them is administered independently. On the other hand riluzole, but not FFA, drastically reduced gasping generation and compromised the ability of mice to autoresucitate. Neither substance P nor forskolin was able to reestablish respiratory activity after riluzole and FFA coapplication. Our results confirm in vitro reports and suggest that eupnea generation in neonates requires a complex neuronal network that includes riluzole- and FFA-sensitive elements and that gasping activity depends mostly on a riluzole-sensitive mechanism.

Epileptiform Activity Induced by Pharmacologic Reduction of M‐Current in the Developing Hippocampus in Vitro

Summary:  Purpose: Benign familial neonatal convulsions (BFNCs), an inheritable epilepsy that occurs in neonates but not in adults, is caused by hypofunctional mutations in genes codifying for the M‐type K+ current. In an attempt to develop an in vitro model of this disease, we tested whether blocking M‐current with linopirdine induces epileptiform activity in brain slices from animals of different ages.