Kaiwen Kam

Distinct Endocytic Pathways Control the Rate and Extent of Synaptic Vesicle Protein Recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.

Excitatory Synaptic Transmission Persists Independently of the Glutamate–Glutamine Cycle

The glutamate–glutamine cycle is thought to be integral in continuously replenishing the neurotransmitter pool of glutamate. Inhibiting glial transfer of glutamine to neurons leads to rapid impairment in physiological and behavioral function; however, the degree to which excitatory synaptic transmission relies on the normal operation of this cycle is unknown. In slices and cultured neurons from rat hippocampus, we enhanced the transfer of glutamine to neurons, a fundamental step in this cycle, by adding exogenous glutamine. Although raising glutamine augments synaptic transmission by increasing vesicular glutamate, access to this synthetic pathway by exogenously applied glutamine to neurons is delayed and slow, challenging mechanisms linking the rapid effects of pharmacological inhibitors to decreased vesicular glutamate. We find that pharmacological inhibitors of glutamine synthetase or system A transporters cause an acute depression of basal synaptic transmission that is rapidly reversible, which is unlikely to be attributable to the rapid loss of vesicular glutamate. Furthermore, release of vesicular glutamate remains robust even during the prolonged removal of glutamine from pure neuronal cultures. We conclude that neurons have the capacity to store or produce glutamate for long periods of time, independently of glia and the glutamate–glutamine cycle.

The peptidergic control circuit for sighing

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

Distinct Inspiratory Rhythm and Pattern Generating Mechanisms in the preBötzinger Complex

In the mammalian respiratory central pattern generator, the preBötzinger complex (preBötC) produces rhythmic bursts that drive inspiratory motor output. Cellular mechanisms initiated by each burst are hypothesized to be necessary to determine the timing of the subsequent burst, playing a critical role in rhythmogenesis. To explore mechanisms relating inspiratory burst generation to rhythmogenesis, we compared preBötC and hypoglossal (XII) nerve motor activity in medullary slices from neonatal mice in conditions where periods between successive inspiratory XII bursts were highly variable and distributed multimodally. This pattern resulted from rhythmic preBötC neural population activity that consisted of bursts, concurrent with XII bursts, intermingled with significantly smaller “burstlets”. Burstlets occurred at regular intervals during significantly longer XII interburst intervals, at times when a XII burst was expected. When a preBötC burst occurred, its high amplitude inspiratory component (I-burst) was preceded by a preinspiratory component that closely resembled the rising phase of burstlets. Cadmium (8 μm) eliminated preBötC and XII bursts, but rhythmic preBötC burstlets persisted. Burstlets and preinspiratory activity were observed in ∼90% of preBötC neurons that were active during I-bursts. When preBötC excitability was raised significantly, burstlets could leak through to motor output in medullary slices and in vivo in adult anesthetized rats. Thus, rhythmic bursting, a fundamental mode of nervous system activity and an essential element of breathing, can be deconstructed into a rhythmogenic process producing low amplitude burstlets and preinspiratory activity that determine timing, and a pattern-generating process producing suprathreshold I-bursts essential for motor output.

Asymmetric control of inspiratory and expiratory phases by excitability in the respiratory network of neonatal mice in vitro

Rhythmic motor behaviours consist of alternating movements, e.g. swing‐stance in stepping, jaw opening and closing during chewing, and inspiration–expiration in breathing, which must be labile in frequency, and in some cases, in the duration of individual phases, to adjust to physiological demands. These movements are the expression of underlying neural circuits whose organization governs the properties of the motor behaviour. To determine if the ability to operate over a broad range of frequencies in respiration is expressed in the rhythm generator, we isolated the kernel of essential respiratory circuits using rhythmically active in vitro slices from neonatal mice. We show respiratory motor output in these slices at very low frequencies (0.008 Hz), well below the typical frequency in vitro (∼0.2 Hz) and in most intact normothermic mammals. Across this broad range of frequencies, inspiratory motor output bursts remained remarkably constant in pattern, i.e. duration, peak amplitude and area. The change in frequency was instead attributable to increased interburst interval, and was largely unaffected by removal of fast inhibitory transmission. Modulation of the frequency was primarily achieved by manipulating extracellular potassium, which significantly affects neuronal excitability. When excitability was lowered to slow down, or in some cases stop, spontaneous rhythm, brief stimulation of the respiratory network with a glutamatergic agonist could evoke (rhythmic) motor output. In slices with slow (<0.02 Hz) spontaneous rhythms, evoked motor output could follow a spontaneous burst at short (≤1 s) or long (∼60 s) intervals. The intensity or timing of stimulation determined the latency to the first evoked burst, with no evidence for a refractory period greater than ∼1 s, even with interburst intervals >60 s. We observed during inspiration a large magnitude (∼0.6 nA) outward current generated by Na+/K+ ATPase that deactivated in 25–100 ms and thus could contribute to burst termination and the latency of evoked bursts but is unlikely to control the interburst interval. We propose that the respiratory network functions over a broad range of frequencies by engaging distinct mechanisms from those controlling inspiratory duration and pattern that specifically govern the interburst interval.

Breathing control center neurons that promote arousal in mice.

A tiny group of neurons in the brainstem matches breathing rate to the level of calmness versus arousal. The calming effect of breathing The rhythmic activity of a cluster of neurons in the brainstem initiates breathing. This cluster is composed of distinct, though intermingled, subgroups of neurons. Yackle et al. found a small, molecularly defined neuronal subpopulation in this breathing rhythm generator that directly projects to a brain center that plays a key role in generalized alertness, attention, and stress (see the Perspective by Sheikbahaei and Smith). Removal of these cells did not affect normal breathing but left the animals unusually calm. The breathing center thus has a direct and dramatic influence on higher-order brain function. Science, this issue p. 1411; see also p. 1370 Slow, controlled breathing has been used for centuries to promote mental calming, and it is used clinically to suppress excessive arousal such as panic attacks. However, the physiological and neural basis of the relationship between breathing and higher-order brain activity is unknown. We found a neuronal subpopulation in the mouse preBötzinger complex (preBötC), the primary breathing rhythm generator, which regulates the balance between calm and arousal behaviors. Conditional, bilateral genetic ablation of the ~175 Cdh9/Dbx1 double-positive preBötC neurons in adult mice left breathing intact but increased calm behaviors and decreased time in aroused states. These neurons project to, synapse on, and positively regulate noradrenergic neurons in the locus coeruleus, a brain center implicated in attention, arousal, and panic that projects throughout the brain.

Emergence of Population Bursts from Simultaneous Activation of Small Subsets of preBötzinger Complex Inspiratory Neurons

During rhythmic movements, central pattern generators (CPGs) trigger bursts of motor activity with precise timing. However, the number of neurons that must be activated within CPGs to generate motor output is unknown. In the mammalian breathing rhythm, a fundamentally important motor behavior, the preBötzinger Complex (preBötC) produces synchronous population-wide bursts of activity to control inspiratory movements. We probed mechanisms underlying inspiratory burst generation in the preBötC using holographic photolysis of caged glutamate in medullary slices from neonatal mice. With stimulation parameters determined to confine photoactivation to targeted neurons, simultaneous excitation of 4–9 targeted neurons could initiate ectopic, endogenous-like bursts with delays averaging 255 ms, placing a critical and novel boundary condition on the microcircuit underlying respiratory rhythmogenesis.

Facing the challenge of mammalian neural microcircuits: taking a few breaths may help

Breathing in mammals is a seemingly straightforward behaviour controlled by the brain. A brainstem nucleus called the preBötzinger Complex sits at the core of the neural circuit generating respiratory rhythm. Despite the discovery of this microcircuit almost 25 years ago, the mechanisms controlling breathing remain elusive. Given the apparent simplicity and well‐defined nature of regulatory breathing behaviour, the identification of much of the circuitry, and the ability to study breathing in vitro as well as in vivo, many neuroscientists and physiologists are surprised that respiratory rhythm generation is still not well understood. Our view is that conventional rhythmogenic mechanisms involving pacemakers, inhibition or bursting are problematic and that simplifying assumptions commonly made for many vertebrate neural circuits ignore consequential detail. We propose that novel emergent mechanisms govern the generation of respiratory rhythm. That a mammalian function as basic as rhythm generation arises from complex and dynamic molecular, synaptic and neuronal interactions within a diverse neural microcircuit highlights the challenges in understanding neural control of mammalian behaviours, many (considerably) more elaborate than breathing. We suggest that the neural circuit controlling breathing is inimitably tractable and may inspire general strategies for elucidating other neural microcircuits.

Practice makes perfect, even for breathing.

Breathing relies on a respiratory rhythm generator. A study characterizes an early emerging oscillatory group of Phox2b-expressing parafacial cells that entrain and couple with the preBotzinger Complex at the onset of fetal breathing.