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Cephalopods produce dynamic colors and skin patterns for communication and camouflage via stratified networks of neuronally actuated yellow, red, and brown chromatophore organs, each filled with thousands of pigment granules. While compositional analysis of chromatophore granules in Doryteuthis pealeii reveals the pigments as ommochromes, the ultrastructural features of the granules and their effects on bulk coloration have not been explored. To investigate this, we isolated granules from specific colored chromatophores and imaged them using multiple modalities. The brown granules are largest with smooth surface coatings. Red granules are intermediate in size with irregular surface textures, and yellow granules are smallest, with rough, porous surfaces. Many of the granules contain sub-granular features that also vary in presentation with color. Correlated light and electron microscopy reveal that differences in hue of individual granules are similarly associated with size, shape, and texture, suggesting that granules may be structurally adapted to modify the dominant visible colors presented within the chromatophores. These findings suggest that granule ultrastructure, not just chemical composition, may be significant in producing the range of colors presented in cephalopod chromatophores., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The visibility of cephalopod chromatophore organs is regulated dynamically by rosettes of obliquely striated radial muscles that dilate or relax the diameter of a central pigmented sacculus in 100-300 ms. Each of the several dozen muscles has a flared proximal end that adheres tightly to its pigmented sacculus and an extremely elongated distal end which branches into single fibrils that anchor into the dermis. This geometry provides ample opportunity for overlap of the many muscles from neighboring chromatophores. The temporal activity of these muscles has been believed to be patterned exclusively by monosynaptic projections from sets of efferent motor axons originating in the chromatophore lobes of the suboesophageal brain. Based on historical observations that distal radial muscles from some chromatophores appear to extend closely to muscles from other chromatophores, we asked whether radial muscles actually make specialized contacts. Using 3D electron microscopy of Doryteuthis pealeii mantle skin, we discovered tight putatively functional muscle-to-muscle contacts between radial muscles from different chromatophores, including elaborate sets of axonal processes located adjacent to those myo-myo junctions. These detailed ultrastructural findings demonstrate auxiliary anatomical routes for radial muscle activation and suggest plausible mechanisms whereby local physical synchronization and axo-axonic processing in the periphery can contribute to chromatophore pattern dynamics such as "passing cloud.", (© 2021 Wiley Periodicals LLC.)

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.

Investigating retinal specializations offers insights into eye functionality. Using retinal wholemount techniques, we investigated the distribution of retinal ganglion cells in the Little skate Leucoraja erinacea by (a) dye-backfilling into the optic nerve prior to retinal wholemounting; (b) Nissl-staining of retinal wholemounts. Retinas were examined for regional specializations (higher numbers) of ganglion cells that would indicate higher visual acuity in those areas. Total ganglion cell number were low compared to other elasmobranchs (backfilled: average 49,713 total ganglion cells, average peak cell density 1,315 ganglion cells mm -2 ; Nissl-stained: average 47,791 total ganglion cells, average peak cell density 1,319 ganglion cells mm -2 ). Ganglion cells fit into three size categories: small (5-20 µm); medium (20-30 µm); large: (≥ 30 µm), and they were not homogeneously distributed across the retina. There was a dorsally located horizontal visual streak with increased ganglion cell density; additionally, there were approximately three local maxima in ganglion cell distribution (potential areae centrales) within this streak in which densities were highest. Using computerized tomography (CT) and micro-CT, geometrical dimensions of the eye were obtained. Combined with ganglion cell distributions, spatial resolving power was determined to be between 1.21 and 1.37 cycles per degree. Additionally, photoreceptor sizes across different retinal areas varied; photoreceptors were longest within the horizontal visual streak. Variations in the locations of retinal specializations appear to be related to the animal's anatomy: shape of the head and eyes, position of eyes, location of tapetum, and shape of pupil, as well as the visual demands associated with lifestyle and habitat type., (© 2018 Wiley Periodicals, Inc.)

The highly diverse and changeable body patterns of cephalopods require the production of whiteness of varying degrees of brightness for their large repertoire of communication and camouflage behaviors. Leucophores are structural reflectors that produce whiteness in cephalopods; they are dermal aggregates of numerous leucocytes containing spherical leucosomes ranging in diameter from 200-2000 nm. In Sepia officinalis leucophores, leucocytes always occur in various combinations with iridocytes, cells containing plates that function as Bragg stacks to reflect light of particular wavelengths. Both spheres and plates contain the high-refractive-index protein reflectin. Four leucophore skin-patterning components were investigated morphologically and with spectrometry. In descending order of brightness they are: white fin spots, White zebra bands, White square, and White head bar. Different densities, thicknesses and proportions of leucocytes and iridocytes were correlated with the relative brightness measurements of the skin. That is, White fin spots and White zebra bands had leucocytes of the highest density, the greatest number of reflective cell layers, and the highest proportion of leucocytes to iridocytes. In contrast, the White square and White head bar had the lowest density of reflective cells, fewer cell layers and the lowest ratios of leucocytes to iridocytes. Leucophores are white in white light, yet reflect whatever colors are in the available light field: e.g. red in red light, green in green light, etc. Leucophores are physiologically passive, thus their ultrastructure alone is capable of diffusing all ambient wavelengths in all directions, regardless of the angle of incident light. However, the specific optical contributions of spherical leucosomes versus the associated plate-like iridosomes in producing whiteness versus brightness are yet to be determined. This study reveals complex morphological arrangements that produce white structural coloration for different brightnesses of skin by differentially combining spheres and plates.

Cuttlefish, Sepia officinalis, possess neurally controlled, pigmented chromatophore organs that allow rapid changes in skin patterning and coloration in response to visual cues. This process of adaptive coloration is enabled by the 500% change in chromatophore surface area during actuation. We report two adaptations that help to explain how colour intensity is maintained in a fully expanded chromatophore when the pigment granules are distributed maximally: (i) pigment layers as thin as three granules that maintain optical effectiveness and (ii) the presence of high-refractive-index proteins-reflectin and crystallin-in granules. The latter discovery, combined with our finding that isolated chromatophore pigment granules fluoresce between 650 and 720 nm, refutes the prevailing hypothesis that cephalopod chromatophores are exclusively pigmentary organs composed solely of ommochromes. Perturbations to granular architecture alter optical properties, illustrating a role for nanostructure in the agile, optical responses of chromatophores. Our results suggest that cephalopod chromatophore pigment granules are more complex than homogeneous clusters of chromogenic pigments. They are luminescent protein nanostructures that facilitate the rapid and sophisticated changes exhibited in dermal pigmentation.

The rapid growth and evolution of the pharmacy profession has created a wide array of opportunities for graduating pharmacists beyond traditional community pharmacy or hospital practice. Management and leadership positions in federal and state healthcare agencies, pharmaceutical companies, hospitals, retail pharmacies, academia and managed care organizations increasingly require the pharmaceutical knowledge obtained through a doctor of pharmacy (PharmD) degree combined with financial, organizational, and management skills. In these innovative positions, pharmacists are being called upon to assume responsibilities as executives and administrators in systems providing pharmacist care services to patients. To endow students with knowledge and skills required to perform the duties required in these decision-making positions, the University of Kentucky College of Pharmacy has established 3 joint degree programs: the PharmD/Master of Business Administration (PharmD/MBA), PharmD/Master of Public Administration (PharmD/MPA), and PharmD/Master of Science in Economics (PharmD/MS). This paper describes these joint degree programs.

An important goal in computational neuroanatomy is the complete and accurate simulation of neuronal morphology. We are developing computational tools to model three-dimensional dendritic structures based on sets of stochastic rules. This paper reports an extensive, quantitative anatomical characterization of simulated motoneurons and Purkinje cells. We used several local and global algorithms implemented in the L-Neuron and ArborVitae programs to generate sets of virtual neurons. Parameters statistics for all algorithms were measured from experimental data, thus providing a compact and consistent description of these morphological classes. We compared the emergent anatomical features of each group of virtual neurons with those of the experimental database in order to gain insights on the plausibility of the model assumptions, potential improvements to the algorithms, and non-trivial relations among morphological parameters. Algorithms mainly based on local constraints (e.g., branch diameter) were successful in reproducing many morphological properties of both motoneurons and Purkinje cells (e.g. total length, asymmetry, number of bifurcations). The addition of global constraints (e.g., trophic factors) improved the angle-dependent emergent characteristics (average Euclidean distance from the soma to the dendritic terminations, dendritic spread). Virtual neurons systematically displayed greater anatomical variability than real cells, suggesting the need for additional constraints in the models. For several emergent anatomical properties, a specific algorithm reproduced the experimental statistics better than the others did. However, relative performances were often reversed for different anatomical properties and/or morphological classes. Thus, combining the strengths of alternative generative models could lead to comprehensive algorithms for the complete and accurate simulation of dendritic morphology.

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure-function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three-dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single-cell neuroanatomy can be characterized quantitatively at several levels. In computer-aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This 'Cartesian' description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise 'blueprint' of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of 'fundamental', measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L-NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the 'computational neuroanatomy' strategy for neuroscience databases.

A technique that transforms a set of images acquired as a rotating frame about an axis (Z axis) into a set of images along the Z axis in presented. This technique is applied to the three-dimensional visualization of the in vivo human lens. A Scheimpfling slit camera acquired 60 optical images through the in vivo human lens. Between each image acquisition the plane containing the slit beam of light was sequentially rotated. This set of 60 images was transformed into a new stack of images on the Z axis. The transformed stack of Z images was visualized with volume rendering software.

An in vivo human lens containing a cataract has been visualized by volume rendering a transformed series of 60 rotated Scheimpflug digital images. The data set was obtained by rotating the Scheimpflug camera about the optic axis of the lens in 3-degree increments. The set of 60 Scheimpflug digital images were mathematically transformed into a new data set in which the images are oriented perpendicular to the optic axis of the eye. The transformed set of optical sections were first aligned to correct for eye movements during the data collection process, then rendered into a three-dimensional volume reconstruction with volume-rendering computer graphics techniques. The viewpoint and the transparency of the volume rendered in vivo human lens were varied in order to observe volume opacities in various regions of the lens. To help visualize lens opacities, the intensity of light scattering was pseudocolor-coded as an integral part of the three-dimensional volume rendering. Three-dimensional, pseudocolored volume rendering of the in vivo human ocular lens represents a new technique to visualize in vivo human cataracts.

We studied thalamocortical afferent (TCA) growth into somatosensory cortex as the whisker barrels emerge in postnatal mice. Ingrowing fibers from the ventrobasal (VB) thalamus were selectively labeled by two means. Under direct vision, individual axons and populations of axons were labeled in vitro with HRP, or in fixed tissue with Dil (1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), in pieces of brain containing both the source nucleus in the thalamus and its cortical target. Many simple thalamocortical afferents are already within the upper cortical plate at birth [postnatal day one (PND1)]. Initially, TCAs from each point in the thalamus distribute in the cortex as two-dimensional "Gaussians," which overlap laterally to constitute a uniform projection pattern. The projection is topographic, because adjacent focal injections within VB label adjacent cortical loci. Subsequent development of barreloids (thalamic representations of the whiskers) partitions the TCA projection into a set of whisker-related Gaussians, centered on cortical targets whose collective topography reflects that of the source pattern. After barreloids form on about PND3, but before barrels appear in cytoarchitecture on about PND5, the overlapping TCAs segregate into dense terminal clusters in layer IV, around which barrels later mature. Time series of single fibers traced with camera lucida explain this transformation that is so noticeable at the population level. As early as PND1, individual TCAs emit multiple ascending collaterals on their horizontal run through white matter and oblique ascent into upper cortex. Subsequently, by PND4, and proceeding at least through PND7, there is accelerated terminal arborization of selected appropriate collateral branches and pruning back of other inappropriate ones. The selection mechanism appears to result from within-group reinforcement events that are stronger for branches toward the center of each whisker-related Gaussian distribution.

Segregation of thalamocortical afferent (TCA) fibers precedes barrel formation in rodent somatosensory cortex (Killackey and Leshin, 1975; Jeanmonod et al., 1981; Jensen and Killackey, 1987b; Senft, 1989; Erzurumlu and Jhaveri, 1990). Hypotheses about the arborization strategies followed by these ingrowing fibers have been generated from evaluation of labeled terminal fragments of mouse TCAs (Senft and Woolsey, 1991a). Those TCAs were of necessity truncated by the histological processing needed to observe them at high resolution. This fragmentation, along with biological variability, forces conclusions about single intact axons to be derived from populations of parts of arbors. To evaluate the hypotheses critically, we designed a computer program to quantify morphological aspects of labeled TCAs drawn with a camera lucida. We constructed algorithms to abstract, from fiber populations, properties minimally affected by truncation. Our program analyzes, and displays as histograms, fiber and branch densities and orientations. To represent these features by additional graphical means, "average" ingrowing TCAs were generated, based on the accumulated statistics of the traced fiber fragments. Quantitative descriptions of TCA populations from postnatal day 1 (PND1) through PND7 are presented. These analyses show that fibers and their branches accumulate with age within the cortical plate (emergent layer IV), and to a lesser extent within developing layer VI. Simultaneously, the distributions of these afferents within cortical laminae transform from uniform to patchy in the plane of the cortex. Peaks exhibit the periodicity typical of mature barrels. Branches become more numerous focally as and where layer IV barrels emerge. Individually traced arbors show reduced total widths consistent with progressive pruning of branches extending into territories of inappropriate barrels, both in layer IV and deeper in the cortex.

Barrels are patterned groups of neurons in rodent somatosensory cortex that correspond one to one with the animal's facial whiskers. Dirichlet domains are a class of convex polygon found frequently in nature, often arising by nucleation from center points. Analytic and graphical methods were devised to verify the hypothesis that Dirichlet domains accurately describe the adult barrel fields of normal mice. We found that normal barrel fields and abnormal barrel fields caused by supernumerary whiskers or lesions to the whisker pad are closely approximated by this mathematical formalism. This implies that each developing cortical barrel organizes about a center point. Experiments in neonatal animals (Senft and Woolsey, 1991a) demonstrate foci in the thalamocortical afferent (TCA) distributions. These results support an hypothesis in which TCAs are the nucleating agents causing barrels to organize as Dirichlet domains. This is made possible because TCA terminals from each barreloid (a whisker-related group of cells in the ventrobasal complex of the thalamus) initially colonize somatosensory cortex with an approximately "Gaussian" distribution. These peaked groups of related TCAs behave as Dirichlet domain centers. They generate barrel structures competitively, in animals with normal or with perturbed whisker patterns, via statistical epigenetic interactions within and between distinct TCA Gaussians associated with separate whiskers. This leads to selective axon outgrowth and pruning of single TCA branches, regulated by the TCA population, and creates beneath each Gaussian the dense knot of related TCA arbors typical of the barrel cortex. Similar parcellation of neuronal processes into contending subgroups having spatially coherent actions could lead to nucleation of other geometric patterns as Dirichlet domains elsewhere in the brain.

The use of high-resolution differential interference contrast(DIC) microscopy on cleared whole-mounts of the circumesophageal nervous system from Hermissenda crassicornis permits visualization of neuronal morphology in detail without the need for physical sectioning. Such optical sectioning, when preceded by intracellular iontophoresis of horseradish peroxidase (HRP) permits rapid and accurate examination of the arborization of electrically characterized neurons. Details such as varicosities and terminal swellings can readily be resolved. This method has revealed new morphological features of neurons implicated in training-specific behavioral modification in Hermissenda, and promises to be of further general use for the quantitative morphometry of electrically identified neurons.