Repeated monitoring of corneal nerves by confocal microscopy as an index of peripheral neuropathy in type-1 diabetic rodents and the effects of topical insulin.

We developed a reliable imaging and quantitative analysis method for in vivo corneal confocal microscopy (CCM) in rodents and used it to determine whether models of type 1 diabetes replicate the depletion of corneal nerves reported in diabetic patients. Quantification was reproducible between observers and stable across repeated time points in two rat strains. Longitudinal studies were performed in normal and streptozotocin (STZ)-diabetic rats, with innervation of plantar paw skin quantified using standard histological methods after 40 weeks of diabetes. Diabetic rats showed an initial increase, then a gradual reduction in occupancy of nerves in the sub-basal plexus so that values were significantly lower at week 40 (68 ± 6%) than age-matched controls (80 ± 2%). No significant loss of stromal or intra-epidermal nerves was detected. In a separate study, insulin was applied daily to the eye of control and STZ-diabetic mice and this treatment prevented depletion of nerves of the sub-basal plexus. Longitudinal studies are viable in rodents using CCM and depletion of distal corneal nerves precedes detectable loss of epidermal nerves in the foot, suggesting that diabetic neuropathy is not length dependent. Loss of insulin-derived neurotrophic support may contribute to the pathogenesis of corneal nerve depletion in type 1 diabetes.

Reward processing abnormalities in Parkinson's disease.

The primary motor cortex is important for motor learning and response selection, functions that require information on the expected and actual outcomes of behavior. Therefore, it should receive signals related to reward. Pathways from reward centers to motor cortex exist in primates. Previously, we showed that gamma aminobutyric acid-A-mediated inhibition in the motor cortex, measured by paired transcranial magnetic stimulation, changes with expectation and uncertainty of money rewards generated by a slot machine simulation. We examined the role of dopamine in this phenomenon by testing 13 mildly affected patients with Parkinson's disease, off and on dopaminergic medications, and 13 healthy, age-matched controls. Consistent with a dopaminergic mechanism, reward expectation or predictability modulated the response to paired transcranial magnetic stimulation in controls, but not in unmedicated patients. A single dose of pramipexole restored this effect of reward, mainly by increasing the paired transcranial magnetic stimulation response amplitude during low expectation. Levodopa produced no such effect. Both pramipexole and levodopa increased risk-taking behavior on the Iowa Gambling Task. However, pramipexole increased risk-taking behavior more in patients showing lower paired transcranial magnetic stimulation response amplitude during low expectation. These results provide evidence that modulation of motor cortex inhibition by reward is mediated by dopamine signaling and that the physiological state of the motor cortex changes with risk-taking tendency in patients on pramipexole. The cortical response to reward expectation may represent an endophenotype for risk-taking behavior in patients on agonist treatment.

Processive flow by biased polymerization mediates the slow axonal transport of actin.

Classic pulse-chase studies have shown that actin is conveyed in slow axonal transport, but the mechanistic basis for this movement is unknown. Recently, we reported that axonal actin was surprisingly dynamic, with focal assembly/disassembly events ("actin hotspots") and elongating polymers along the axon shaft ("actin trails"). Using a combination of live imaging, superresolution microscopy, and modeling, in this study, we explore how these dynamic structures can lead to processive transport of actin. We found relatively more actin trails elongated anterogradely as well as an overall slow, anterogradely biased flow of actin in axon shafts. Starting with first principles of monomer/filament assembly and incorporating imaging data, we generated a quantitative model simulating axonal hotspots and trails. Our simulations predict that the axonal actin dynamics indeed lead to a slow anterogradely biased flow of the population. Collectively, the data point to a surprising scenario where local assembly and biased polymerization generate the slow axonal transport of actin without involvement of microtubules (MTs) or MT-based motors. Mechanistically distinct from polymer sliding, this might be a general strategy to convey highly dynamic cytoskeletal cargoes.

Axonal actin in action: Imaging actin dynamics in neurons.

Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

A dynamic formin-dependent deep F-actin network in axons.

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal "actin hotspots" along axons-spaced ∼3-4 µm apart-where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons-a phenomenon we call "actin trails." Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal "actin rings" described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin-but not Arp2/3-dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable "actin rings" providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.