The locus coeruleus directs sensory-motor reflex amplitude across environmental contexts.

Purposeful movement across unpredictable environments requires quick, accurate, and contextually appropriate motor corrections in response to disruptions in balance and posture.1,2,3 These responses must respect both the current position and limitations of the body, as well as the surrounding environment,4,5,6 and involve a combination of segmental reflexes in the spinal cord, vestibulospinal and reticulospinal pathways in the brainstem, and forebrain structures such as the motor cortex.7,8,9,10 These motor plans can be heavily influenced by the animal's surrounding environment, even when that environment has no mechanical influence on the perturbation itself. This environmental influence has been considered as cortical in nature, priming motor responses to a perturbation.8,11 Similarly, postural responses can be influenced by environments that alter threat levels in humans.12,13,14,15,16,17,18 Such studies are generally in agreement with work done in the mouse showing that optogenetic stimulation of the lateral vestibular nucleus (LVN) only results in motor responses when the animal is on a balance beam at height and not when walking on the stable surface of a treadmill.10 In general, this ability to flexibly modify postural responses across terrains and environmental conditions is a critically important component of the balance system.19,20 Here we show that LVN-generated motor corrections can be altered by manipulating the surrounding environment. Furthermore, environmental influence on corrections requires noradrenergic signaling from the locus coeruleus, suggesting a potential link between forebrain structures that convey sensory information about the environment and brainstem circuits that generate motor corrections.

Pitx2 cholinergic interneurons are the source of C bouton synapses on brainstem motor neurons.

Cholinergic neuromodulation has been described throughout the brain and has been implicated in various functions including attention, food intake and response to stress. Cholinergic modulation is also thought to be important for regulating motor systems, as revealed by studies of large cholinergic synapses on spinal motor neurons, called C boutons, which seem to control motor neuron excitability in a task-dependent manner. C boutons on spinal motor neurons stem from spinal interneurons that express the transcription factor Pitx2. C boutons have also been identified on the motor neurons of specific cranial nuclei. However, the source and roles of cranial C boutons are less clear. Previous studies suggest that they originate from Pitx2+ and Pitx2- neurons, in contrast to spinal cord C boutons that originate solely from Pitx2 neurons. Here, we address this controversy using mouse genetics, and demonstrate that brainstem C boutons are Pitx2+ derived. We also identify new Pitx2 populations and map the cholinergic Pitx2 neurons of the mouse brain. Taken together, our data present important new information about the anatomical organization of cholinergic systems which impact motor systems of the brainstem. These findings will enable further analyses of the specific roles of cholinergic modulation in motor control.

Arrays of microscopic organic LEDs for high-resolution optogenetics.

Optogenetics is a paradigm-changing new method to study and manipulate the behavior of cells with light. Following major advances of the used genetic constructs over the last decade, the light sources required for optogenetic control are now receiving increased attention. We report a novel optogenetic illumination platform based on high-density arrays of microscopic organic light-emitting diodes (OLEDs). Because of the small dimensions of each array element (6 × 9 µm(2)) and the use of ultrathin device encapsulation, these arrays enable illumination of cells with unprecedented spatiotemporal resolution. We show that adherent eukaryotic cells readily proliferate on these arrays, and we demonstrate specific light-induced control of the ionic current across the membrane of individual live cells expressing different optogenetic constructs. Our work paves the way for the use of OLEDs for cell-specific optogenetic control in cultured neuronal networks and for acute brain slices, or as implants in vivo.

Adenosine-mediated modulation of ventral horn interneurons and spinal motoneurons in neonatal mice.

Neuromodulation allows neural networks to adapt to varying environmental and biomechanical demands. Purinergic signaling is known to be an important modulatory system in many parts of the CNS, including motor control circuitry. We have recently shown that adenosine modulates the output of mammalian spinal locomotor control circuitry (Witts EC, Panetta KM, Miles GB. J Neurophysiol 107: 1925-1934, 2012). Here we investigated the cellular mechanisms underlying this adenosine-mediated modulation. Whole cell patch-clamp recordings were performed on ventral horn interneurons and motoneurons within in vitro mouse spinal cord slice preparations. We found that adenosine hyperpolarized interneurons and reduced the frequency and amplitude of synaptic inputs to interneurons. Both effects were blocked by the A1-type adenosine receptor antagonist DPCPX. Analysis of miniature postsynaptic currents recorded from interneurons revealed that adenosine reduced their frequency but not amplitude, suggesting that adenosine acts on presynaptic receptors to modulate synaptic transmission. In contrast to interneurons, recordings from motoneurons revealed an adenosine-mediated depolarization. The frequency and amplitude of synaptic inputs to motoneurons were again reduced by adenosine, but we saw no effect on miniature postsynaptic currents. Again these effects on motoneurons were blocked by DPCPX. Taken together, these results demonstrate differential effects of adenosine, acting via A1 receptors, in the mouse spinal cord. Adenosine has a general inhibitory action on ventral horn interneurons while potentially maintaining motoneuron excitability. This may allow for adaptation of the locomotor pattern generated by interneuronal networks while helping to ensure the maintenance of overall motor output.

Anatomy and function of cholinergic C bouton inputs to motor neurons.

Motor control circuitry of the central nervous system must be flexible so that motor behaviours can be adapted to suit the varying demands of different states, developmental stages, and environments. Flexibility in motor control is largely provided by neuromodulatory systems which can adjust the output of motor circuits by modulating the properties and connectivity of neurons within them. The spinal circuitry which controls locomotion is subject to a range of neuromodulatory influences, including some which are intrinsic to the spinal cord. One such intrinsic neuromodulatory system, for which a wealth of anatomical information has recently been combined with new physiological data, is the C bouton system. C boutons are large, cholinergic inputs to motor neurons which were first described over 40 years ago but whose source and function have until recently remained a mystery. In this review we discuss how the convergence of anatomical, molecular genetic and physiological data has recently led to significant advances in our understanding of this unique neuromodulatory system. We also highlight evidence that C boutons are involved in spinal cord injury and disease, revealing their potential as targets for novel therapeutic strategies.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.

Glial-derived adenosine modulates spinal motor networks in mice.

The activation of purinergic receptors modulates central pattern generators controlling rhythmic motor behaviors, including respiration in rodents and swimming in frog tadpoles. The present study aimed to determine whether purinergic signaling also modulates the mammalian locomotor central pattern generator. This was investigated by using isolated spinal cord preparations obtained from neonatal mice in which locomotor-related activity can be induced pharmacologically. The application of either ATP or adenosine led to a reduction in the frequency of locomotor activity recorded from ventral roots. ATP had no effect when applied in the presence of both the adenosine receptor antagonist theophylline and the ectonucleotidase inhibitor ARL67156, demonstrating that the effects of ATP application result from the breakdown of ATP to adenosine and subsequent activation of adenosine receptors. The application of theophylline or the A(1)-specific antagonist cyclopentyl dipropylxanthine, but not the A(2A)-receptor antagonist SCH58261, caused an increase in locomotor burst frequency, demonstrating that endogenously derived adenosine activates A(1) receptors during locomotor network activity. Furthermore, theophylline had no effect in the presence of the ectonucleotidase inhibitor ARL67156 or the glial toxins methionine sulfoximine or ethyl fluoracetate, suggesting that endogenous adenosine is derived from ATP, which is released from glia. Finally, adenosine had no effect on slow rhythmic activity recorded upon blockade of all inhibitory transmission, suggesting that adenosine may act via the modulation of inhibitory transmission. Together, these data highlight endogenous purinergic gliotransmission, involving activation of A(1) receptors, as an important intrinsic modulatory system controlling the frequency of activity generated by spinal locomotor circuitry in mammals.