Passive vs. active touch-induced activity in the developing whisker pathway.

The mouse trigeminal (V) system undergoes significant postnatal structural and functional developmental changes. Histological modules (barrelettes, barreloids and barrels) in the brainstem, thalamus and cortex related to actively moved (whisking) tactile hairs (vibrissae) on the face allow detailed studies of development. High-resolution [(3) H]2-deoxyglucose (2DG) emulsion autoradiography with cytochrome oxidase histochemistry was used to analyze neuronal activity changes related to specific whisker modules in the developing and mature mouse V system provoked by passive (experimenter-induced) and active (animal-induced) displacements of a single whisker (D4). We tested the hypothesis that neuronal activity patterns change in relation to the onset of active touch (whisking) on postnatal day (P)14. Quantitative image analyses revealed: (i) on P7, when whisker-like patterns of modules are clear, heightened 2DG activity in all appropriate modules in the brainstem, thalamus and cortex; (ii) on P14, a transitory activity pattern coincident with the emergence of whisking behavior that presages (iii) strong labeling of the spinal V subnucleus interpolaris and barrel cortex produced by single-whisker-mediated active touch in adults and (iv) at all above-listed ages and structures, significant suppression of baseline activity in some modules surrounding those representing the stimulated whisker. Differences in activity patterns before and after the onset of whisking behavior may be caused by neuronal activity induced by whisking, and by strengthening of modulatory projections that alter the activity of subcortical inputs produced by whisking behavior during active touch.

Review on Cowan WM, Gottlieb DI, Hendrickson AE, Price JL, Woolsey TA. 1972. The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res 37: 21-51.

UNLABELLED: Axoplasmically transported proteins synthesized in neuronal somata labeled by radioactively labeled amino acids (tritium), following local targeted injections for tracing of pathways in the central nervous system using autoradiography. Results from a number of neuronal systems, including: the rat olfactory bulb; cortico-thalamic projections in the mouse; commissural connections of the rat hippocampus; and retinal projections in the monkey and chick are documented. Pathway origins are clear, as the number and distribution of the labeled cells and the normal structure of the injection site is preserved. Light and electron microscopic autoradiography shows that proteins are transported, at two rates: rapid transport (>100mm/day) of fewer proteins accumulating in axon terminals; and, slow transport (1-5mm/day) of the bulk of labeled proteins distributed along the length of axons. Different survival times can be selected to evaluate terminal projection field(s) or pathways from origin to termination. The clarity of autoradiographic labeling of pathways and their terminations is comparable to other techniques (such as the Nauta-Gygax and the Fink-Heimer methods and the electron microscopy of terminal degeneration). Labeled amino acids do not label molecules in fibers of passage and there is no retrograde transport of labeled material from the axon terminals. The functional polarity of fiber pathways can be easily established. We summarize the merits of this technique is based upon an established physiological properties of neurons that are summarized in contrast to currently used techniques dependent upon pathological changes in neurons, axons, or axonal terminals. ARTICLE ABSTRACT: This article considers a heavily cited Brain Research article that reported an extremely important turning point in the ability to demonstrate neuroanatomical pathways in the central nervous system. Using radioactive leucine microinjections into the brain, neurons synthesized proteins from this amino acid that were transported down their axons to the terminal synapses on the target neurons. Tracing the transport of the labeled protein by autoradiography permitted quantitative analysis of projections and pathways. As a result, pathway analysis was transformed from studying the degenerating processes of lesioned neurons to the study of intact pathways in non-manipulated brains. The classical protocol has since been widely applied and used to investigate countless brain circuits. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Re: Woolsey TA, van der Loos H. 1970. The structural organization of layer IV in the somatosensory region (S I) of mouse cerebral cortex. Brain Res. 17: 205-242.

UNLABELLED: Axoplasmically transported proteins synthesized in neuronal somata labeled by radioactively labeled amino acids (tritium), following local targeted injections for tracing of pathways in the central nervous system using autoradiography. Results from a number of neuronal systems, including: the rat olfactory bulb; cortico-thalamic projections in the mouse; commissural connections of the rat hippocampus; and retinal projections in the monkey and chick are documented. Pathway origins are clear, as the number and distribution of the labeled cells and the normal structure of the injection site is preserved. Light and electron microscopic autoradiography shows that proteins are transported, at two rates: rapid transport (>100mm/day) of fewer proteins accumulating in axon terminals; and, slow transport (1-5mm/day) of the bulk of labeled proteins distributed along the length of axons. Different survival times can be selected to evaluate terminal projection field(s) or pathways from origin to termination. The clarity of autoradiographic labeling of pathways and their terminations is comparable to other techniques (such as the Nauta-Gygax and the Fink-Heimer methods and the electron microscopy of terminal degeneration). Labeled amino acids do not label molecules in fibers of passage and there is no retrograde transport of labeled material from the axon terminals. The functional polarity of fiber pathways can be easily established. We summarize the merits of this technique is based upon an established physiological properties of neurons that are summarized in contrast to currently used techniques dependent upon pathological changes in neurons, axons, or axonal terminals. ABSTRACT: The cytoarchitecture of layer IV in mouse SmI cerebral cortex was examined in.formalin-fixed, Nissl-stained and Cox-fixed, Golgi-Nissl-stained sections cut coronally and tangentially to the pia, A multicellular cytoarchitectonic unit is described in layer IV, roughly cylindrical, 100-400um in diameter, and perpendicular to the pia. Because of their characteristic shape we call these structures barrels. Each barrel is a ring of neurons, the side, which surrounds a less cellular hollow. The nearly acellular reigion surrounding each barrel and separating adjacent barrels is the septum. Barrels are discussed in relation to observations reported in several earlier papers on the mouse cortex. The barrel field (all barrels) has remarkable constancy by all measures: from one hemisphere to the next and from one specimen to the next. A consistent part of the barrel field is the postero-medial barrel subield (PMBSF). Barrels in the PMBSF are larger, elliptical in shape, organized into five distinct rows and their numbers are constant. It is postulated that each barrel in the PMBSF is the cortical correlate of a contralateral mystacial vibrissa (whisker). On the basis of counts of barrels and of all facial sinus hairs a 'one barrel-one vibrissa' hypothesis is proposed. The general hypothesis is that barrels are the morphological manifestation in layer IV of the functional cortical columns discovered by physiologists. The barrels offer excellent opportunities for integrated studies of sensory cerebral cortex at a degree of resolution previously not possible. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Evaluation to Improve a High School Summer Science Outreach Program.

The goal of the Young Scientist Program (YSP) at Washington University School of Medicine in St. Louis (WUSM) is to broaden science literacy and recruit talent for the scientific future. In particular, YSP seeks to expose underrepresented minority high school students from St. Louis public schools (SLPS) to a wide variety of careers in the sciences. The centerpiece of YSP, the Summer Focus Program (SFP), is a nine-week, intensive research experience for competitively chosen rising high school seniors (Scholars). Scholars are paired with volunteer graduate student, medical student, or postdoctoral fellow mentors who are active members of the practicing scientific community and serve as guides and exemplars of scientific careers. The SFP seeks to increase the number of underrepresented minority students pursuing STEM undergraduate degrees by making the Scholars more comfortable with science and science literacy. The data presented here provide results of the objective, quick, and simple methods developed by YSP to assess the efficacy of the SFP from 2006 to 2013. We demonstrate that the SFP successfully used formative evaluation to continuously improve the various activities within the SFP over the course of several years and in turn enhance student experiences within the SFP. Additionally we show that the SFP effectively broadened confidence in science literacy among participating high school students and successfully graduated a high percentage of students who went on to pursue science, technology, engineering, and mathematics (STEM) majors at the undergraduate level.

Spatial integration of vascular changes with neural activity in mouse cortex.

The authors evaluated representations of discretely activated, neighboring brain regions using real-time optical intrinsic signals by transcranial imaging with 540-nm and 610-nm broadband illumination of the mouse barrel cortex. Iron filings were glued to two neighboring whiskers (C2 + D2) that were stimulated magnetically, singly and together. Real-time images were collected, averaged, and analyzed statistically. Postmortem filling of arteries with fluorescent beads was shown in relation to histochemical staining of barrels to accurately relate surface changes to functional cortical columns. Significant optical intrinsic signal changes are related to overlapping distributions of arterioles that feed the two separate areas. Activation of adjacent and interacting cortical columns leads not only to increased magnitude of vascular responses in those columns, but also to wider spatial extent of absorption changes occurring principally in areas of cortex fed by vessels upstream of the active cortex. The localization of changing hemoglobin absorption around upstream blood vessels and their vascular domains suggests that propagated vasodilation of upstream parent vessels is greater when vasodilatory signals from separate areas of active cortex converge on common arterioles that feed them.

A method to measure the effective spread of focally injected muscimol into the central nervous system with electrophysiology and light microscopy.

A method was developed to quantitate the volume of brain inactivated by muscimol focally injected. Tritiated muscimol was injected into the cerebellum and closely spaced sequential microelectrode recordings made at different depths by penetrations in an X-Y pattern centered at the injection site to evaluate changes in spontaneous activity in the tissue volume. Animals were euthanized after survivals from 40 min to 6 h, the cerebellum sectioned in the sagittal plane, and the sections dried onto glass slides. The slides were dipped in photographic emulsion, exposed in the dark and developed. Silver grain densities were quantitated by light microscopy from measured standards. The extent and concentration of bound, labeled muscimol co-varied with the observed reduction in recorded spontaneous activity. For future studies, the distribution and density of silver grains alone can serve as an accurate spatial indicator of the area of muscimol inactivation at high spatial resolution.

In vivo imaging in a murine model of glioblastoma.

OBJECTIVE: To use in vivo imaging methods in mice to quantify intracranial glioma growth, to correlate images and histopathological findings, to explore tumor marker specificity, to assess effects on cortical function, and to monitor effects of chemotherapy. METHODS: Mice with DBT glioma cell tumors implanted intracranially were imaged serially with a 4.7-T small-animal magnetic resonance imaging (MRI) scanner. MRI tumor volumes were measured and correlated with postmortem histological findings. Different nonspecific and specific positron emission tomography radiopharmaceuticals, [18F]2-fluoro-2-deoxy-d-glucose, [18F]3'-deoxy-3'-fluorothymidine, or [11C]RHM-I, a sigma2-receptor ligand, were visualized with microPET (CTI-Concorde MicroSystems LLC, Knoxville, TN). Intrinsic optical signals were imaged serially during contralateral whisker stimulation to study the impact of tumor growth on cortical function. Other groups of mice were imaged serially with MRI after one or two doses of the antimitotic N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). RESULTS: MRI and histological tumor volumes were highly correlated (r2 = 0.85). Significant binding of [11C]RHM-I was observed in growing tumors. Over time, tumors reduced and displaced (P # 0.001) whisker-activated intrinsic optical signals but did not change intrinsic optical signals in the contralateral hemisphere. Tumor growth was delayed 7 days after a single dose of BCNU and 18 days after two doses of BCNU. Mean tumor volume 15 days after DBT implantation was significantly smaller for treated mice (1- and 2-dose BCNU) compared with controls (P = 0.0026). CONCLUSION: Mouse MRI, positron emission tomography, and optical imaging provide quantitative and qualitative in vivo assessments of intracranial tumors that correlate directly with tumor histological findings. The combined imaging approach provides powerful multimodality assessments of tumor progression, effects on brain function, and responses to therapy.

Computational hyperspectral interferometry for studies of brain function: proof of concept.

Hyperspectral interferometric microscopy uses a unique combination of optics and algorithm design to extract information. Local brain activity rapidly changes local blood flow and red blood cell concentration (absorption) and oxygenation (color). We demonstrate that brain activity evoked during whisker stimulation can be detected with hyperspectral interferometric microscopy to identify the active whisker-barrel cortex in the rat brain. Information about constituent components is extracted across the entire spectral band. Algorithms can be flexibly optimized to discover, detect, quantify, and visualize a wide range of significant biological events, including changes relevant to the diagnosis and treatment of disease.

Glutamate receptor blockade alters the development of intracortical connections in rat barrel cortex.

We tested the hypothesis that glutamate receptor mediated activity is required for the postnatal development of intracortical connections in layers II/III of rodent barrel cortex. To block glutamate receptors, a slow release polymer (elvax) loaded with a glutamate receptor antagonist (D-AP5) was targeted subdurally over the future rat barrel cortex on P0 (day of birth). On P14-16 biotinylated dextran amine (BDA) was injected under the elvax into all layers to label neurons retrogradely. A BDA injection was made stereotactically at the mirror site of the untreated hemisphere of each animal. The animals survived to P22-24. Injection sites and retrogradely labeled cell bodies were identified in tangential sections in relation to the barrel map. D-AP5 treated and untreated hemispheres were matched according to the location of the injection site in the barrel map. Glutamate receptor blockade did not prevent the growth of intrinsic projections, but altered their organization. The normal row-like asymmetry of connections in untreated hemispheres was lacking in the D-AP5 treated cortex (ANOVA, p=0.02). Cortical activity mediated through glutamate receptors contributes to the correct development of connections between barrel columns in layers II/III.

Albumin-binding MR blood pool agents as MRI contrast agents in an intracranial mouse glioma model.

Intravenous MRI contrast agents are commonly used to improve the detection of intracranial tumors and other central nervous system (CNS) lesions for diagnosis and treatment planning. Two small-molecule, albumin-binding blood pool contrast agents (MP-2269 and MS-325) of potential clinical significance were evaluated at 1.5 Tesla in a mouse glioma model and compared with an extracellular contrast agent (OptiMARK). Tumor image contrast was significantly enhanced and long-lived following administration of 30 micromole/kg of the blood pool agents: specifically, contrast enhancement peaked slowly at 25-30 min following administration, remained constant for >3 hr, and returned to baseline within 20 hr. Comparable but "transient" enhancement was achieved using 100 micromole/kg OptiMARK: specifically, contrast enhancement peaked rapidly at 2-5 min following administration and then declined over 40 min. The blood pool contrast agents demonstrated an approximately threefold increased dose-effectiveness and a lengthened window of tumor contrast enhancement in comparison to commonly available extracellular contrast agents. This demonstrates the potential of alternative contrast-enhanced (CE) MRI examination protocols for tumor detection.