Optimization of direct cortical stimulation using tibial versus median nerve sensory mapping during midline brain tumor resection: illustrative case.

BACKGROUND: During brain tumor resection, neurophysiological mapping and monitoring help surgeons locate, characterize, and functionally assess eloquent brain areas in real time. The selection of mapping and monitoring targets has implications for successful surgery. Here, the authors compare direct cortical stimulation (DCS) as suggested by median nerve (MN) with posterior tibial nerve (PTN) cortical sensory mapping (SM) during mesial lesion resection. OBSERVATIONS: Recordings from a 6-contact cortical strip served to generate an MN and a PTN sensory map, which indicated the strip was anterior to the central sulcus. Responses exhibited an amplitude gradient with no phase reversal (PR). DCS, elicited through a stimulus probe or contact(s) of the strip, yielded larger responses from the corresponding sensory mapped limb; that is, PTN SM resulted in larger lower limb muscle responses than those suggested by MN SM. LESSONS: SM of the MN and PTN is effective for localizing eloquent cortical areas wherein the PTN is favored in surgery for mesial cortical tumors. The recorded amplitude of the cortical somatosensory evoked potential is a valuable criterion for defining the optimal location for DCS, despite an absent PR. The pathway at risk dictates the specifics of SM, which subsequently defines the optimal location for DCS.

Non-additive stabilization by halogenated amino acids reveals protein plasticity on a sub-angstrom scale.

It has been a long-standing goal to understand the structure-stability relationship of proteins, as optimal stability is essential for protein function and highly desirable for protein therapeutics. Halogenation has emerged as a minimally invasive strategy to probe the physical characteristics of proteins in solution, as well as enhance the structural stabilities of proteins for therapeutic applications. Although advances in synthetic chemistry and genetic code expansion have allowed for the rapid synthesis of proteins with diverse chemical sequences, much remains to be learned regarding the impact of these mutations on their structural integrity. In this contribution, we present a systematic study of three well-folded model protein systems, in which their structural stabilities are assessed in response to various hydrogen-to-halogen atom mutations. Halogenation allows for the perturbation of proteins on a sub-angstrom scale, offering unprecedented precision of protein engineering. The thermodynamic results from these model systems reveal that in certain cases, proteins can display modest steric tolerance to halogenation, yielding non-additive consequences to protein stability. The observed sub-angstrom sensitivity of protein stability highlights the delicate arrangement of a folded protein core structure. The stability data of various halogenated proteins presented herein should also provide guidelines for using halogenation as a strategy to improve the stability of protein therapeutics.

Exploring and exploiting polar-π interactions with fluorinated aromatic amino acids.

Fluorination has become an increasingly attractive strategy in protein engineering for both basic research and biomedical applications. Thus researchers would like to understand the consequences of fluorination to the structure, stability, and function of target proteins. Although a substantial amount of work has focused on understanding the properties of fluorinated aliphatic amino acids, much less is known about fluorinated aromatic residues. In addition, polar-π interactions, often referred to as aromatic interactions, may play a significant role in protein folding and protein-protein interactions. Fluorination of aromatic residues presents an ideal strategy for probing polar-π interactions in proteins. This Account summarizes the recent studies of the incorporation of fluorinated aromatic amino acids into proteins. Herein we discuss the effects of fluorinating aromatic residues and rationalize them in the context of polar-π interactions. The results strongly support the proposal that polar-π interactions are energetically significant to protein folding and function. For example, an edge-face interaction of a pair of phenylalanines contributes as much as -1 kcal/mol to protein stability, while cation-π interactions can be much stronger. Furthermore, this new knowledge provides guidelines for protein engineering with fluorination. Importantly, incorporating perfluorinated aromatic residues into proteins enables novel mechanisms of molecular recognition that do not exist in native proteins, such as arene-perfluoroarene stacking. Such novel mechanisms can be used for programming protein folding specificity and engineering peptide-based materials.

Experimental evaluation of CH-π interactions in a protein core.

CH-π stacks up! Using the protein α(2) D as a model system, we estimate that a CH-π contact between cyclohexylalanine (Cha) and phenylalanine (F) contributes approximately -0.7 kcal mol(-1) to the protein stability. The stacking F-Cha pairs are sequestered in the core of the protein, where water interference does not exist (see figure). Therefore, the observed energetic gain should represent the inherent magnitude and upper limit of the CH-π interactions.

Novel iboga alkaloid congeners block nicotinic receptors and reduce drug self-administration.

18-Methoxycoronaridine, a novel iboga alkaloid congener, reduces drug self-administration in animal models of addiction. Previously, we proposed that these effects are mediated by the ability of 18-methoxycoronaridine to inhibit nicotinic alpha3beta4 acetylcholine receptors. In an attempt to identify more potent 18-methoxycoronaridine analogs, we have tested a series of 18-methoxycoronaridine congeners by whole-cell patch clamp recording of HEK 293 cells expressing recombinant nicotinic alpha3beta4 receptors or glutamate NR1/NR2B N-methyl-d-aspartate (NMDA) receptors. The congeners exhibited a range of inhibitory potencies at alpha3beta4 receptors. Five congeners had IC(50) values similar to 18-methoxycoronaridine, and all of these were ineffective at NMDA receptors. The congeners also retained their ability to reduce morphine and methamphetamine self-administration. These data are consistent with the importance of nicotinic alpha3beta4 receptors as a therapeutic target to modulate drug seeking. These compounds may constitute a new class of synthetic agents that act via the nicotinic alpha3beta4 mechanism to combat addiction.

Intracellular injection in fixed slices: obtaining complete dendritic arbors of large cells.

Intracellular injection of Lucifer yellow into fixed brain slices is widely used to demonstrate dendritic morphology. A major limitation of this technique is that large dendritic arbors are usually truncated at the cut surfaces. Here we describe modifications that allowed us to obtain complete dendritic arbors of large spiny stellate cells. Lucifer Yellow cadaverine biotin-X (LY-X) was injected into individual neurons within 300-1000 microm thick aldehyde-fixed slices of kitten visual cortex. Subsequently, the LY-X was histochemically reacted using standard ABC methods to obtain a permanent record of the injected cells. Dendrites, studded with a variety of dendritic spines, were darkly labeled and well defined against virtually no background. Somatic spines, dendritic varicosities and growth cones were common in the younger animals. Computer-assisted reconstructions demonstrated that, in older animals, the dendritic arbors of cells injected in 300 microm slices were truncated, whereas the arbors of cells injected deep within thick slices were complete. The modifications described here remove the most critical limitation of intracellular injection in slices, allowing quantitative analysis of even large dendritic arbors.

Rapid automated three-dimensional tracing of neurons from confocal image stacks.

Algorithms are presented for fully automatic three-dimensional (3-D) tracing of neurons that are imaged by fluorescence confocal microscopy. Unlike previous voxel-based skeletonization methods, the present approach works by recursively following the neuronal topology, using a set of 4 x N2 directional kernels (e.g., N = 32), guided by a generalized 3-D cylinder model. This method extends our prior work on exploratory tracing of retinal vasculature to 3-D space. Since the centerlines are of primary interest, the 3-D extension can be accomplished by four rather than six sets of kernels. Additional modifications, such as dynamic adaptation of the correlation kernels, and adaptive step size estimation, were introduced for achieving robustness to photon noise, varying contrast, and apparent discontinuity and/or hollowness of structures. The end product is a labeling of all somas present, graph-theoretic representations of all dendritic/axonal structures, and image statistics such as soma volume and centroid, soma interconnectivity, the longest branch, and lengths of all graph branches originating from a soma. This method is able to work directly with unprocessed confocal images, without expensive deconvolution or other preprocessing. It is much faster that skeletonization, typically consuming less than a minute to trace a 70-MB image on a 500-MHz computer. These properties make it attractive for large-scale automated tissue studies that require rapid on-line image analysis, such as high-throughput neurobiology/angiogenesis assays, and initiatives such as the Human Brain Project.