Jan-Marino Ramirez

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing

Cell type–specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps

Are different forms of breathing derived from one or multiple neural networks? We demonstrate that brainstem slices containing the pre-Bötzinger complex generated two rhythms when normally oxygenated, with striking similarities to eupneic (‘normal’) respiration and sighs. Sighs were triggered by eupneic bursts under control conditions, but not in the presence of strychnine (1 μM). Although all neurons received synaptic inputs during both activities, the calcium channel blocker cadmium (4 μM) selectively abolished sighs. In anoxia, sighs ceased, and eupneic activity was reconfigured into gasping, which like eupnea was insensitive to 4 μM cadmium. This reconfiguration was accompanied by suppression of synaptic inhibition. We conclude that a single medullary network underlies multiple breathing patterns.

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.

Selective lesioning of the cat pre‐Bötzinger complex in vivo eliminates breathing but not gasping

1 To examine the functional importance of the pre‐Bötzinger complex for breathing we micro‐injected, under in vivo conditions, the calcium channel blocker ω‐conotoxin GVIA and the sodium channel blocker tetrodotoxin (TTX) into the ventrolateral medulla of adult cats, while monitoring respiratory rhythmic motor output in the phrenic nerve. 2 ω‐Conotoxin GVIA caused a highly localized synaptic ablation by blocking presynaptic N‐type calcium channels. When injecting 5–60 fmol ω‐conotoxin GVIA unilaterally, the amplitude of phrenic nerve activity decreased bilaterally and sometimes disappeared, indicating central apnoea. These effects were reversible and could only be induced in a very localized area of the pre‐Bötzinger complex. By injecting ω‐conotoxin GVIA several times during an experiment and analysing the areas where injections affected respiratory activity, it was possible to map exactly the anatomical extent of the area critical for respiratory rhythm generation. 3 Following the precise localization of the pre‐Bötzinger complex with ω‐conotoxin GVIA, we injected TTX to induce an irreversible inactivation of this region. TTX injected unilaterally into the pre‐Bötzinger complex irreversibly reduced the amplitude of phrenic nerve activity. Bilateral TTX injections eliminated respiratory rhythmic activity, causing a persistent central apnoea. 4 After bilateral lesioning of the pre‐Bötzinger complex, it was still possible to induce gasping during hypoxia or asphyxia, indicating that respiration and gasping are generated by two different neuronal networks. 5 We propose that ω‐conotoxin GVIA as employed in this study to investigate the functional role of the pre‐Bötzinger complex can also be used as a general pharmacological approach to map other neuronal networks. We call this the ‘ω‐conotoxin GVIA tracing’ method.

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.

The neuronal mechanisms of respiratory rhythm generation

New, improved in vivo and in vitro approaches have led to a better understanding of the mechanisms that generate respiratory rhythm, which depends on a complex interaction between network and intrinsic membrane properties. The pre-Bötzinger complex in the ventrolateral medulla is particularly important for respiratory rhythm generation. This complex can be studied in isolation, and it contains all the known types of respiratory neurons that are now amenable to detailed cellular and molecular analyses.

Entrainment of the locomotor rhythm by group Ib afferents from ankle extensor muscles in spinal cats

Summary1. Previous studies have concluded that the timing of the locomotor rhythm can be strongly influenced by input from group Ib afferents from leg extensor muscles (Duysens and Pearson 1980; Conway et al. 1987). The main objective of the present study was to obtain additional evidence for this conclusion by examining the characteristics of entrainment of the locomotor rhythm by rhythmic stimulation of group I afferents and by rhythmic force pulses in the ankle extensor muscles. 2. A reduced, non-immobilized preparation was developed in spinal cats that allowed isometric contractions of ankle extensor muscles to be elicited by ventral root stimulation during the expression of locomotor activity. The same preparation was used to examine the influence of electrically stimulating group I afferents from the ankle extensors and the effect of rhythmically stretching these muscles. The locomotor rhythm was initiated by sustained mechanical stimulation of the perineum following the administration of Clonidine and, in some preparations, Naloxone. 3. The timing of the onset of flexor burst activity was examined during entrainment with saw-tooth and ramp-and-hold stretches of the ankle extensor muscles. Flexor bursts were initiated about 200 ms following the release from the stretch, and this latency was independent of the entrainment frequency. 4. The locomotor rhythm was readily entrained by rhythmic contractions of the ankle extensor muscles produced by ventral root stimulation provided the magnitude of the contractions was greater than about 10N. Repetitive stimulation of group I muscle afferents from the ankle extensors also entrained the locomotor rhythm, with the timing of motor activity being similar to that during entrainment with rhythmic muscle contractions. Burst activity in the ipsilateral extensors was coincident with the stimulus trains in both cases. This similarity argues for entrainment being produced mainly by input from group Ib afferents. 5. The functional implication of the results of this and previous studies is that input from group Ib afferents during the stance phase of walking acts to inhibit generation of flexor burst activity and to promote extensor activity. The proposal that a decline in Ib activity near the end of the stance phase is involved in regulating the stance to swing transition is discussed.

Neuromodulation and the orchestration of the respiratory rhythm.

The respiratory system is continuously modulated by numerous aminergic and peptidergic substances that act at all levels of integration: from the sensory level to the level of central networks and motor nuclei. The same neuronal networks receive inputs from multiple modulators released locally as well as from distal nuclei. All parameters of respiratory control are controlled by multiple neuromodulators. By partly converging onto similar G-proteins and second messenger systems, acetylcholine, norepinephrine, histamine, serotonin (5-HT), dopamine, ATP, substance P, cholecystokinin (CCK) can increase frequency, regularity and amplitude of respiratory activity. Yet, the same modulator can also exert differential effects on respiratory activity by acting on different receptors partly in the same neurons. In the pre-Bötzinger complex (pre-BötC) modulators can differentially modulate frequency and amplitude in different types of pacemaker neurons. Similarly motoneurons located in different motor nuclei receive differential amplitude modulation from different modulators. Thus, modulators are capable of orchestrating and modulating different parameters of respiratory activity by differentially targeting different cellular targets. A disturbance in modulatory control may lead to Sudden Infant Death Syndrome (SIDS) and erratic breathing.

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.