K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve

The localization of Shaker-type K⁺ channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic β-subunit Kvβ2 were colocalized in juxtaparanodal zones. During development, clustering of K⁺ channels lagged behind that for nodal Na⁺ channels by about 5 days. In contrast to the PNS, K⁺ channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K⁺ channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na⁺ channel clusters, and little or no detectable clustering of K⁺ channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K⁺ channel clustering and localization.     

Context dependency of a limb withdrawal reflex in the caterpillar Manduca sexta

The proleg withdrawal reflex in the caterpillar Manduca sexta is a robust, well-characterized system for investigating the integration of sensory information with centrally patterned behavior. The reflex is evoked by stimulating mechanosensory hairs--planta hairs--located at the tip of each proleg. We studied the expression of this reflex by combining video recordings and electromyographic recordings from the main retractor muscles of the proleg, the principal and accessory planta retractor muscles. In intact animals, the nature of the response depended on the motor context of the animal. Animals which were standing quietly showed great variability in both the kinematic properties of proleg withdrawal, and the corresponding muscle electrical activity. Animals which were hanging upside down from a wooden dowel exhibited a much faster reflex, with retraction of the proleg occurring slightly faster than in standing animals, but re-extension of the proleg to the substrate being considerably faster. In crawling animals, expression of the reflex depended on the phase of the crawling cycle during which stimulation occurred. The reflex in a given proleg was suppressed during stance phase of that proleg. During swing phase, however, planta hair stimulation evoked proleg withdrawal, resulting in an assistance reflex. In contrast. isolated abdomens showed much less variability in the reflex. A comparison of the relationship between retractor muscle activity and the resulting proleg movement showed significant correlations between both the duration of activity and the number of muscle spikes, and the size of the associated proleg withdrawal. This is a promising system in which to investigate how central neuronal circuits accomplish context-dependency of motor behavior.     

Reciprocal Changes in Phosphorylation and Methylation of Mammalian Brain Sodium Channels in Response to Seizures

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.     

Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.     

Modeling locomotion of a soft-bodied arthropod using inverse dynamics

Most bio-inspired robots have been based on animals with jointed, stiff skeletons. There is now an increasing interest in mimicking the robust performance of animals in natural environments by incorporating compliant materials into the locomotory system. However, the mechanics of moving, highly conformable structures are particularly difficult to predict. This paper proposes a planar, extensible-link model for the soft-bodied tobacco hornworm caterpillar, Manduca sexta, to provide insight for biologists and engineers studying locomotion by highly deformable animals and caterpillar-like robots. Using inverse dynamics to process experimentally acquired point-tracking data, ground reaction forces and internal forces were determined for a crawling caterpillar. Computed ground reaction forces were compared to experimental data to validate the model. The results show that a system of linked extendable joints can faithfully describe the general form and magnitude of the contact forces produced by a crawling caterpillar. Furthermore, the model can be used to compute internal forces that cannot be measured experimentally. It is predicted that between different body segments in stance phase the body is mostly kept in tension and that compression only occurs during the swing phase when the prolegs release their grip. This finding supports a recently proposed mechanism for locomotion by soft animals in which the substrate transfers compressive forces from one part of the body to another (the environmental skeleton) thereby minimizing the need for hydrostatic stiffening. The model also provides a new means to characterize and test control strategies used in caterpillar crawling and soft robot locomotion.