Dimiter Prodanov

Spatial clustering analysis in neuroanatomy: Applications of different approaches to motor nerve fiber distribution

Spatial organization of the nerve fibers in the peripheral nerves may be important for the studies of axonal regeneration, the degenerative nerve diseases and the construction of interfaces with peripheral nerves, such as nerve prostheses. Functional topography of motor axons related to the gastrocnemius muscle was revealed in the ventral spinal roots by retrograde tracing. Gastrocnemius muscles of adult rats were injected with the tracer Fluoro-Gold. After 3 days of survival the animals were sacrificed and their ventral roots were harvested, sectioned, and imaged on a fluorescence microscope. Maps of the traced fibers were automatically analyzed using a novel approach, local spatial clustering statistics, that tested for occurrences of clusters of motor fibers and visualized them. Thresholds indicating the presence of clustering at various scales of observation were computed based on series of Monte Carlo simulations of random spatial point patterns. Clusters were visualized by kernel interpolation. The approach was tested on simulated data and subsequently applied to the motor fiber maps in the ventral roots. Results revealed clustering of the motor fibers innervating the gastrocnemius muscle at the level of the L6 ventral spinal root. The analysis was validated using Voronoi tessellation and nearest neighbor analysis.

Automatic morphometry of synaptic boutons of cultured cells using granulometric analysis of digital images.

Numbers, linear density, and surface area of synaptic boutons can be important parameters in studies on synaptic plasticity in cultured neurons. We present a method for automatic identification and morphometry of boutons based on filtering of digital images using granulometric analysis. Cultures of cortical neurons (DIV8 and DIV21) were fixed and marked with fluorescently labeled antibodies for synapsin I (a marker for synaptic boutons) and MAP-2 (a marker for dendrites). Images were acquired on a confocal microscope and automatically processed. Granulometry, a morphological operator sensitive to the geometry and size of objects, was used to construct a filter passing fuzzy fluorescent grains of a certain size. Next, the filter was overlaid with the original image (masking) and the positive pixels were identified by an integral intensity threshold (thresholding). Disjoint grains, representing individual boutons, were reconstructed from the connected pixels above the threshold, numbered and their area was measured. In total, 1498 boutons with a mean diameter of 1.63 +/- 0.49 microm (S.D.) were measured. Comparisons with manual counts showed that the proposed method was capable of identifying boutons in a systematic manner at the light microscopic level and was a viable alternative to manual bouton counting.

Three-dimensional topography of the motor endplates of the rat gastrocnemius muscle

Spatial distribution of motor endplates affects the shape of the electrical activity recorded from muscle. In order to provide information for realistic models of action potential propagation within muscles, we assembled three‐dimensional maps of the motor endplates of the rat medial gastrocnemius (MGM) and lateral gastrocnemius (MGL) muscles. The maps were assembled from histological cross sections stained for acetylcholinesterase activity. Within MGL, the motor endplates formed three columns along its longitudinal axis. Within MGM, the motor endplates were arranged in a leaf‐like body that shifted obliquely from proximal to distal. As inferred from the proximo‐distal distribution of the cross‐sectional projection area, the majority of the motor endplates were concentrated in the middle of MGL and in the distal third of MGM. Regions of maximal motor endplate concentration are considered most suitable for injections of neuroactive substances, such as neuronal tracers. The assembled maps of the gastrocnemius muscles can be used as guides for such injections within the motor endplate zones. Muscle Nerve, 2005

A post-ischaemic single administration of galanthamine, a cholinesterase inhibitor, improves learning ability in rats.

Transient forebrain ischaemia is widely observed in clinical practice. We have examined the effect of a single administration of the Cholinesterase inhibitor galanthamine (2 mgkg−1, i.p.) 25 min after reperfusion in male Sprague‐Dawley rats (180 ± 20 g) after a 20‐min common carotid artery occlusion.

And Then There Was Light: Perspectives of Optogenetics for Deep Brain Stimulation and Neuromodulation

Deep Brain Stimulation (DBS) has evolved into a well-accepted add-on treatment for patients with severe Parkinsons disease as well as for other chronic neurological conditions. The focal action of electrical stimulation can yield better responses and it exposes the patient to fewer side effects compared to pharmaceuticals distributed throughout the body toward the brain. On the other hand, the current practice of DBS is hampered by the relatively coarse level of neuromodulation achieved. Optogenetics, in contrast, offers the perspective of much more selective actions on the various physiological structures, provided that the stimulated cells are rendered sensitive to the action of light. Optogenetics has experienced tremendous progress since its first in vivo applications about 10 years ago. Recent advancements of viral vector technology for gene transfer substantially reduce vector-associated cytotoxicity and immune responses. This brings about the possibility to transfer this technology into the clinic as a possible alternative to DBS and neuromodulation. New paths could be opened toward a rich panel of clinical applications. Some technical issues still limit the long term use in humans but realistic perspectives quickly emerge. Despite a rapid accumulation of observations about patho-physiological mechanisms, it is still mostly serendipity and empiric adjustments that dictate clinical practice while more efficient logically designed interventions remain rather exceptional. Interestingly, it is also very much the neuro technology developed around optogenetics that offers the most promising tools to fill in the existing knowledge gaps about brain function in health and disease. The present review examines Parkinsons disease and refractory epilepsy as use cases for possible optogenetic stimulation therapies.