The changing landscape of membrane protein structural biology through developments in electron microscopy

Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins

Supraspinal control of locomotion

International audienceLocomotion is a basic motor function generated and controlled by genetically defined neuronal networks. The pattern of muscle synergies is generated in the spinal cord, whereas neural centers located above the spinal cord in the brainstem and the forebrain are essential for initiating and controlling locomotor movements. One such locomotor control center in the brainstem is the mesencephalic locomotor region (MLR), first discovered in cats and later found in all vertebrate species tested to date. Over the last years, we have investigated the cellular mechanisms by which this locomotor region operates in lampreys. The lamprey MLR is a well-circumscribed region located at the junction between the midbrain and hindbrain. Stimulation of the MLR induces locomotion with an intensity that increases with the stimulation strength. Glutamatergic and cholinergic monosynaptic inputs from the MLR are responsible for excitation of reticulospinal (RS) cells that in turn activate the spinal locomotor networks. The inputs are larger in the rostral than in the caudal hindbrain RS cells. MLR stimulation on one side elicits symmetrical excitatory inputs in RS cells on both sides, and this is linked to bilateral projections of the MLR to RS cells. In addition to its inputs to RS cells, the MLR activates a well-defined group of muscarinoceptive cells in the brainstem that feeds back strong excitation to RS cells in order to amplify the locomotor output. Finally, the MLR gates sensory inputs to the brainstem through a muscarinic mechanism. It appears therefore that the MLR not only controls locomotor activity but also filters sensory influx during locomotion

Serotoninergic modulation of sensory transmission to brainstem reticulospinal cells

Sensory inputs are subjected to modulation by central neural networks involved in controlling movements. It has been shown that serotonin (5‐HT) modulates sensory transmission. This study examines in lampreys the effects of 5‐HT on sensory transmission to brainstem reticulospinal (RS) neurons and the distribution of 5‐HT cells that innervate RS cells. Cells were recorded intracellularly in the in vitro isolated brainstem of larval lampreys. Trigeminal nerve stimulation elicited disynaptic excitatory responses in RS neurons, and bath application of 5‐HT reduced the response amplitude with maximum effect at 10 μm. Local ejection of 5‐HT either onto the RS cells or onto the relay cells decreased sensory‐evoked excitatory postsynaptic potentials (EPSPs) in RS cells. The monosynaptic EPSPs elicited from stimulation of the relay cells were also reduced by 5‐HT. The reduction was maintained after blocking either N‐methyl‐d‐aspartate (NMDA) or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptors. The local ejection of glutamate over RS cells elicited excitatory responses that were only slightly depressed by 5‐HT. In addition, 5‐HT increased the threshold for eliciting sustained depolarizations in response to trigeminal nerve stimulation but did not prevent them. Combined 5‐HT immunofluorescence with axonal tracing revealed that the 5‐HT innervation of RS neurons of the middle rhombencephalic reticular nucleus comes mainly from neurons in the isthmic region, but also from neurons located in the pretectum and caudal rhombencephalon. Our results indicate that 5‐HT modulates sensory transmission to lamprey brainstem RS cells

Morphology and connectivity of parabrachial and cortical inputs to gustatory thalamus in rats

The ventroposterior medialis parvocellularis (VPMpc) nucleus of the thalamus, the thalamic relay nucleus for gustatory sensation, receives primary input from the parabrachial nucleus, and projects to the insular cortex. To reveal the unique properties of the gustatory thalamus in comparison with archetypical sensory relay nuclei, this study examines the morphology of synaptic circuitry in the VPMpc, focusing on parabrachiothalamic driver input and corticothalamic feedback. Anterogradely visualized parabrachiothalamic fibers in the VPMpc bear large swellings. At electron microscope resolution, parabrachiothalamic axons are myelinated and make large boutons, forming multiple asymmetric, adherent, and perforated synapses onto large‐caliber dendrites and dendrite initial segments. Labeled boutons contain dense‐core vesicles, and they resemble a population of terminals within the VPMpc containing calcitonin gene‐related peptide. As is typical of primary inputs to other thalamic nuclei, parabrachiothalamic terminals are over five times larger than other inputs, while constituting only 2% of all synapses. Glomeruli and triadic arrangements, characteristic features of other sensory thalamic nuclei, are not encountered. As revealed by anterograde tracer injections into the insular cortex, corticothalamic projections in the VPMpc form a dense network of fine fibers bearing small boutons. Corticothalamic terminals within the VPMpc were also observed to synapse on cells that were retrogradely filled from the same injections. The results constitute an initial survey describing unique anatomical properties of the rodent gustatory thalamus. J. Comp. Neurol. 523:139–161, 2015. © 2014 Wiley Periodicals, Inc. Using biotinylated tract tracers and light and electron microscopy, the authors provide quantitative ultrastructural characterization of two inputs that arrive to the gustatory thalamic nucleus (ventroposterior medialis parvocellularis nucleus [VPMpc]): parabrachiothalamic axons that bring the primary input, and corticothalamic axons that provide the feedback input.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109654/1/cne23673.pd

PHOSPHENE IMAGES OF THALAMIC SLEEP RHYTHMS INDUCED BY SELF-HYPNOSIS

A medical writer describes internally-generated light sensations (phosphenes) induced by a technique of self-hypnosis that combines relaxation, convergent eye movement, and attentive fixation. The phosphene images include: (1) a threshold sequence of receding annuli, (2) amorphous phosphene mists or clouds, and (3) phosphene clouds with two levels of brightness and color saturation. These images share some similarities with visions of light reported by religious mystics. Based on an analysis of the distinctive spatiotemporal characteristics exhibited by the phosphenes. the author proposes the hypothesis that they are generated by thalamic sleep rhythms oscillating in the lateral geniculate nucleus (LGN). Since humans usually lose consciousness at the onset of non-rapid-eye-movement sleep (NREMS), the author also proposes the hypothesis that his technique of phosphene induction preserves consciousness, despite the operation of thalamic sleep rhythms, because eye movements and attentive fixation send excitatory feedback to the visual pathways. This selective facilitation of visual neurons appears to preserve their signal-processing capacity even though synchronous sleep rhythms may be installed in the non-visual thalamus. The author speculates that this selective disruption of sleep rhythm activity in the visual pathways may be the mechanism that produces the cutaneous analgesia (hypnoanalgesia) he experiences when he induces phosphenes

PHOSPHENE IMAGES OF THALAMIC SLEEP RHYTHMS INDUCED BY SELF-HYPNOSIS

A medical writer describes internally-generated light sensations (phosphenes) induced by a technique of self-hypnosis that combines relaxation, convergent eye movement, and attentive fixation. The phosphene images include: (1) a threshold sequence of receding annuli, (2) amorphous phosphene mists or clouds, and (3) phosphene clouds with two levels of brightness and color saturation. These images share some similarities with visions of light reported by religious mystics. Based on an analysis of the distinctive spatiotemporal characteristics exhibited by the phosphenes. the author proposes the hypothesis that they are generated by thalamic sleep rhythms oscillating in the lateral geniculate nucleus (LGN). Since humans usually lose consciousness at the onset of non-rapid-eye-movement sleep (NREMS), the author also proposes the hypothesis that his technique of phosphene induction preserves consciousness, despite the operation of thalamic sleep rhythms, because eye movements and attentive fixation send excitatory feedback to the visual pathways. This selective facilitation of visual neurons appears to preserve their signal-processing capacity even though synchronous sleep rhythms may be installed in the non-visual thalamus. The author speculates that this selective disruption of sleep rhythm activity in the visual pathways may be the mechanism that produces the cutaneous analgesia (hypnoanalgesia) he experiences when he induces phosphenes

Sensory Activation of Command Cells for Locomotion and Modulatory Mechanisms: Lessons from Lampreys

Sensorimotor transformation is one of the most fundamental and ubiquitous functions of the central nervous system. Although the general organization of the locomotor neural circuitry is relatively well understood, less is known about its activation by sensory inputs and its modulation. Utilizing the lamprey model, a detailed understanding of sensorimotor integration in vertebrates is emerging. In this article, we explore how the vertebrate central nervous system integrates sensory signals to generate motor behavior by examining the pathways and neural mechanisms involved in the transformation of cutaneous and olfactory inputs into motor output in the lamprey. We then review how 5-HT acts on these systems by modulating both sensory inputs and motor output. A comprehensive review of this fundamental topic should provide a useful framework in the fields of motor control, sensorimotor integration and neuromodulation

CELLULAR AND CIRCUIT PROPERTIES OF SLOW OSCILLATIONS IN THE THALAMIC RETICULAR NUCLEUS

During sleep, neurons in the thalamic reticular nucleus (TRN) generate distinct types of oscillatory activity. While the reciprocal synaptic circuits between TRN and sensory thalamic nuclei underlie the generation of sleep spindles, the mechanisms regulating slow (\u3c1 \u3eHz) forms of thalamic oscillations are poorly understood. Under in vitro conditions, in the absence of synaptic inputs, TRN neurons can generate slow oscillations in a cell-intrinsic manner. Activation of postsynaptic Group 1 metabotropic glutamate receptors (mGluR) leads to long-lasting plateau potentials thought to be mediated by both T-type calcium currents and calcium-activated nonselective cation currents (ICAN). However, the identity of ICAN and the possible contribution of thalamic circuits to slow rhythmic activity remain unclear. Using intracellular recordings of neurons in thalamic slices derived from adult male and female mice, I recorded slow forms of rhythmic activity in TRN neurons. Slow oscillations were driven by fast glutamatergic inputs from thalamic relay neurons, but did not require postsynaptic mGluR activation. For a significant minority of TRN neurons (33%), synaptic inputs or brief depolarizing current steps led to plateau potentials and persistent firing (PF), and in turn, resulted in persistent synaptic inhibition in postsynaptic relay neurons of the ventrobasal thalamus (VB). Pharmacological approaches indicated that plateau potentials were triggered by calcium influx through T-type calcium channels and mediated by calcium and voltage-dependent transient receptor potential melastatin 4 (TRPM4) channels. Taken together, my results suggest that thalamic circuits can generate slow oscillatory activity, mediated by an interplay of TRN-VB synaptic circuits that generate rhythmicity and TRN cell-intrinsic mechanisms that control PF and oscillation frequency

Investigations into the organisation and morphology of vagal preganglionic neurones and the anatomical identification of the chemistry and origin of some of their synaptic inputs

The investigations in this thesis involve the use of neuroanatomical techniques in the study of vagal preganglionic neurones (VPNs) in the medulla oblongata of the rat and cat. Light microscopic examination of VPNs identified by the injection of horseradish peroxidase (HRP) into the cervical vagus nerve of the rat revealed that they lay mainly in two areas of the medulla - the dorsal vagal nucleus (DVN) and the nucleus ambiguus (NA). Labelled cells in the DVN (median diameter 18jim) were arranged in a tightly packed group. In contrast, labelled neurones in the NA were arranged in two identifiable groups. The most dorsal of these groups consisted of tightly packed cells with median diameter of 30μm (compact group - cNA) whereas the other group were smaller (median D 25μm) and were scattered in ventrolateral regions of the medulla (vINA). Neurones in each group were identified as being statistically different in diameter and soma area from those in other groups (Student's t-test). Cardiac vagal preganglionic neurones (CVPNs), retrogradely labelled by the injection of cholera toxin-HRP into the right atrium, were located mainly in the vINA with median soma diameter 25μm. Ultrastructural examination of the groups of VPNs revealed that they shared some morphological characteristics. VPNs in the NA, particularly those in the vINA, were observed to be intermingled with neurones retrogradely labelled from the phrenic motor nucleus in the spinal cord and were often morphologically indistinguishable. VPNs in the NA were also intermingled with neuropeptide Y immunoreactive neurones. The chemistry of inputs to VPNs was examined using a combination of retrograde tracing to identify VPNs, immunocytochemistry to detect various neurotransmitter chemicals, and electron microscopy. Serotonin, substance P and neuropeptide Y were identified in boutons forming asymmetric type synaptic contacts with VPNs in the nucleus ambiguus of the rat which were retrogradely labelled from the heart or the cervical vagus nerve. The origin of inputs to VPNs was investigated by the ionophoretic injection of HRP into regions of the cat ventral medulla where antidromic potentials were recorded to stimulation of the cervical vagus nerve. Retrogradely labelled cells were localised both contralaterally and ipsilateral ly in the nucleus tractus solitarius, raphe nuclei, parabrachial nucleus, periaqueductal gray matter and the NA. To determine if the NTS was a source of synaptic input to VPNs in the NA, VPNs were identified by retrograde tracing followed by the ionophoretic injection of the anterograde tracer biocytin into regions of the NTS where evoked potentials were recorded to stimulation of the carotid sinus or cervical vagus nerve. Light microscopic examination revealed anterogradely labelled boutons and fibres in the NA, some of which were in close association with retrogradely labelled VPNs. Subsequent electron microscopic examination revealed some of these boutons form synaptic specialisations with retrogradely labelled VPNs

Les mécanismes synaptiques et intrinsèques qui sous-tendent l’activité des cellules réticulospinales (RS) en réponse à une stimulation sensorielle de type cutané chez la lamproie

Chez diverses espèces animales, les informations sensorielles peuvent déclencher la locomotion. Ceci nécessite l’intégration des informations sensorielles par le système nerveux central. Chez la lamproie, les réseaux locomoteurs spinaux sont activés et contrôlés par les cellules réticulospinales (RS), système descendant le plus important. Ces cellules reçoivent des informations variées provenant notamment de la périphérie. Une fois activées par une brève stimulation cutanée d’intensité suffisante, les cellules RS produisent des dépolarisations soutenues de durées variées impliquant des propriétés intrinsèques calcium-dépendantes et associées à l’induction de la nage de fuite. Au cours de ce doctorat, nous avons voulu savoir si les afférences synaptiques ont une influence sur la durée des dépolarisations soutenues et si l’ensemble des cellules RS partagent des propriétés d’intégration similaires, impliquant possiblement les réserves de calcium internes. Dans un premier temps, nous montrons pour la première fois qu’en plus de dépendre des propriétés intrinsèques des cellules réticulospinales, les dépolarisations soutenues dépendent des afférences excitatrices glutamatergiques, incluant les afférences spinales, pour perdurer pendant de longues périodes de temps. Les afférences cutanées ne participent pas au maintien des dépolarisations soutenues et les afférences inhibitrices glycinergique et GABAergiques ne sont pas suffisantes pour les arrêter. Dans un deuxième temps, nous montrons que suite à une stimulation cutanée, l’ensemble des cellules RS localisées dans les quatre noyaux réticulés possèdent un patron d’activation similaire et elles peuvent toutes produire des dépolarisations soutenues dont le maintien ne dépend pas des réserves de calcium internes. Enfin, les résultats obtenus durant ce doctorat ont permis de mieux comprendre les mécanismes cellulaires par lesquels l’ensemble des cellules RS intègrent une brève information sensorielle et la transforment en une réponse soutenue associée à une commande motrice.In various animal species, sensory information can initiate locomotion. This relies on the integration of sensory inputs by the central nervous system. In lampreys, the spinal locomotor networks are activated and controlled by the reticulospinal cells (RS) which constitute the main descending system. In turn, RS cells receive information coming from various synaptic inputs such as the sensory afferents. Once activated by a brief cutaneous stimulation of sufficient strength, RS cells display sustained depolarizations of various durations that rely on calcium-dependant intrinsic properties and lead to the onset of escape swimming. During the course of this Ph.D, we aimed at determining whether synaptic inputs can modulate the duration of the sustained depolarizations and if the different populations of RS cells share the same integrative properties, possibly involving the internal calcium stores. First, our results show for the first time that excitatory glutamatergic inputs, including ascending spinal feedback, contribute to prolong the sustained depolarizations for long periods of time. Cutaneous inputs do not contribute to maintain the sustained depolarizations and inhibitory glycinergic and GABAergic inputs are not sufficient to stop them. Second, we show that in response to cutaneous stimulation, the RS located in the four reticular nuclei display a similar activation pattern and can all produce sustained depolarizations which do not depend on internal calcium release to be maintained. Finally, the results obtained during this Ph.D allowed us to better understand the cellular mechanisms by which the RS cells integrate and transform a brief sensory information into a sustained response associated with a motor command