Thomas A. Woolsey

Localized dynamic changes in cortical blood flow with whisker stimulation corresponds to matched vascular and neuronal architecture of rat barrels.

The hypothesis that functional groups of neurons in whisker barrels are linked to a modular organization of cortical vessels was tested. Endovascular casts demonstrated cortical capillary networks resembling the whisker barrel pattern that were fed from the middle cerebral artery. In histological sections, dense capillaries apparently were confined to single barrels and were supplied by one or a few penetrating arterioles. The barrel field in cortical layer IV was localized in relation to surface arteriovenous patterns. Living vessels were imaged through a closed cranial window under anesthesia with a fluorescence microscope and SIT or ICCD cameras. After intracarotid injections of fluorescein isothiocyanate–dextrans, saline, or 3 μm latex beads, changes in arteriolar diameter, arteriovenous transit times (AVTTs), and bead velocities were measured. When row C whiskers were stroked at 4–5 Hz for 1 min, blood flow increased in arterioles that supplied contralateral row C barrels as demonstrated by postmortem histology. AVTTs slowed significantly in vessels supplying adjacent cortex. We hypothesize that cerebral vascular units supply individual whisker barrels and are functionally linked to them for precise focal regulation of cerebral blood flow.

An On-Line Digital-Computer System for the Semiautomatic Analysis of Golgi-Impregnated Neurons

Rapid and accurate measurements of neuronal processes in Golgi preparations are possible with the aid of a small computer (Glaser and Van der Loos, 1965). This paper describes a system for doing this, using a small digital computer which controls stepping motors (0.5-?m steps) attached to the stage (x, y axes) and fine focus (z axis) of a microscope. The observer tracks the processes, topological information, such as the location of the soma, dendritic origins, branch points, and ends of processes, is signaled to the computer by special controls, and the x, y, and z coordinates of each are stored in digital format. The computer printout yields 1) individual x, y, and z coordinates with associated topological identifiers; 2) quantitative data for each dendritic segment in order (i.e., primary and secondary branches, etc.); and 3) computes the actual linear dimensions of each segment in microns. An associated oscilloscope display can 1) display the whole neuron, or individual processes, by connecting recorded points by vectors; 2) identify each dendrite; 3) rotate (continuously or by a specified angle) the whole cell, or individual dendrites, around any selected point or axis; and 4) indicate spatial relationships by dynamic rotation, intensity modulation, or stereo pairs. The performance of this system has been evaluated for accuracy and the repeatability of measurements.

Infraorbital nerve blockade from birth does not disrupt central trigeminal pattern formation in the rat

We tested the hypothesis that patterned primary afferent impulse activity during early postnatal periods is necessary for central trigeminal pattern formation. Newborn rats had their whiskers trimmed daily and new slices of slow release polymer containing the sodium channel blocker, tetrodotoxin, were placed under the infraorbital nerve every 8 h for up to 9 days. Electrophysiological recordings indicated that trigeminal ganglion cells were unresponsive to peripheral stimuli and chronically silenced. Trigeminal ganglion cell numbers were unaffected by nerve blockade. Cytochrome oxidase staining patterns in the trigeminal brainstem complex, thalamus, and barrel cortex were normal on postnatal day 1, 3, 5, 7, or 9 (n = 4 each). Whisker-related patches were of normal sizes and staining densities. Similar negative results were obtained in 9 rats in which whiskers were trimmed daily and the long-acting local anesthetic bupivacaine was injected into the whisker pad at 2.5- to 4-h intervals from birth to sacrifice on postnatal day 5-9. Cytochrome oxidase staining patterns and patch properties again did not differ from normal. Thus, trigeminal pattern formation occurs even when the entire infraorbital nerve is silenced from birth.

Cytoarchitectonic correlates of the vibrissae in the medullary trigeminal complex of the mouse

Cytoarchitectonic patterns in the medullary trigeminal complex of the mouse corresponding to mystacial vibrissae are described. These patterns are found within trigeminal sub-nuclei principalis, interpolaris and caudalis. The patterns are due to differential cell packing and are homeomorphic to the arrangement of the mystacial vibrissae on the face. The cytoarchitecture is similar, but complementary, to patterns of trigeminal afferents previously described using histochemical staining methods. Neonatal cautery of groups of vibrissae produces appropriate, specific and localized cytoarchitectural changes within all 3 trigeminal sub-nuclei.

Functional organization of mouse and rat SmI barrel cortex following vibrissal damage on different postnatal days.

This study was undertaken to determine the functional properties of neurons in the anatomically altered somatosensory cortex after neonatal whisker damage. In mice and rats neonatal lesions of the facial vibrissae change the anatomical organization of barrels in the contralateral SmI cortex. These changes depend on the pattern and severity of the peripheral damage and the developmental age of the animals. To understand some of the functional correlates of these anatomical changes, the middle row of vibrissae (row C) was damaged in mice on postnatal days 1, 3, and 5 and in rats on postnatal days 1 and 5. The receptive field properties of single cortical units were studied after the animals matured. In 24 mice and 15 rats a total of 1,370 units were characterized in microelectrode penetrations which passed through the somatosensory cortex either tangential or perpendicular to the pia. Units were localized anatomically with respect to both barrel and laminar boundaries, and the extent of the peripheral damage was assessed histologically. The data revealed an orderly representation of the sensory periphery that coincided with the altered cytoarchitectonic organization of the SmI cortex. Specifically: (1) Units in the enlarged row B or row D barrels responded primarily to row B or row D whiskers. (2) In layer IV, units in the altered row C cortex either could not be reliably driven from the periphery, were activated by stimulation of scar tissue in the damaged facial row C, or were driven by adjacent, intact row B or row D whiskers. (3) Units in supra- and infragranular layers either had no row C representation or incorporated scar tissue in their receptive fields in a topographically correct fashion. Responses of units to stimulation of scar tissue were qualitatively similar to those elicited from intact vibrissae, which also activated them. (4) In SmII, units that responded to whiskers had receptive fields whose organization matched the representation of the periphery observed in SmI. (5) There was no mapping of nonmystacial pad structures in the barrel cortex, and there were no units with abnormal multiwhisker interactions when laminar boundaries were taken into account. These data indicate that neonatal damage to the whiskers alters both the anatomical arrangement of the barrels and the physiologically determined somatotopic representation of the sensory periphery in a parallel and predictable fashion.

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.

Local axonal trajectories in mouse barrel cortex

SummaryQuantitative studies were made of the distribution of labeled intracortical axons after focal injections of horseradish peroxidase (HRP) into mouse barrel cortex, in vitro. The pattern of labeled fibers was compared to that of labeled cell bodies with respect to the barrel map in layer IV. We analyzed 4 cortices with injections in supragranular layers and centered above a single barrel row. Computer microscope/image analysis routines were used to collect the data and to perform various statistical analyses on them. The distributions of both labeled cells and fibers in layer IV and in the infragranular layers show strong connectional tendencies between barrels representing a whisker row. This result is consistent with single unit recordings from barrel cortex. Fiber labeling is more widespread than cell body labeling in layer IV. In addition, the fibers show a directional bias into the adjacent anterior barrel row (e.g., C → D, D → E). In earlier 2-deoxyglucose (2-DG) studies of behaving animals, the anterior barrel rows were more heavily labeled; inter-row projections are therefore predominantly from less active to more active barrel columns. These data show that labeled fiber distribution differs from the distribution pattern of labeled cell bodies. The findings indicate that integration of information between whisker rows within barrel cortex involves asymmetrical connections within layer IV and infragranular layers.

Blood flow in single surface arterioles and venules on the mouse somatosensory cortex measured with videomicroscopy, fluorescent dextrans, nonoccluding fluorescent beads, and computer-assisted image analysis.

Cortical surface vessels were monitored through closed cranial windows with an epifluorescence microscope and SIT or ICCD cameras. Fluorescent dextrans or 1.3 μm latex beads were injected into the contralateral jugular vein for plasma labeling and for vascular transits. For close arterial transits, these tracers or physiological saline were injected into the ipsilateral external carotid artery. AVTTs were calculated from intensity differences of tracers between a branch of the MCA and a vein draining the same cortical region over time. AVTTs for saline dilutions of RBCs were significantly shorter (0.73 times) than for dextrans. Both dextrans and beads distributed with plasma. With FITC–dextran, inner diameters of arterioles and venules averaged 6 μm larger than hemoglobin under green light. This difference was likely due to the segregation of red blood cells and plasma during flow. Velocities of individual fluorescent beads were measured in pial vessels by strobe epi-illumination. Plots of bead velocities against radial position in arterioles were blunted parabolas. Peak shear rates in the marginal layer next to the vessel walls were determined directly from bead tracks in arterioles (D = 21–71 μm) and were 1.32 times the Poiseuille estimate. The calculated peak wall shear stress was 39 ± 14 dyn/cm2 (mean ± SD) for these arterioles but was probably severalfold greater in the smallest terminal pial arterioles. Vmax near the axes of arterioles increased with D+0.5. The calculated peak wall shear rate was highest in small arterioles and decreased with D−0.5. The calculated flow Q increased with D+2.5. These methods permit direct, simultaneous, dynamic measurements on multiple identified cerebral microvessels.

Templates for locating the whisker area in fresh flattened mouse and rat cortex

A way was devised to locate the projection fields (barrels) of the whiskers in fresh neocortex of rats and mice. The cortex was quickly removed and flattened prior to fixation and staining for cytochrome oxidase. The barrels were mapped in relation to the flattened cortex by superimposing specimen and vascular outlines. The topographic location of the barrel field is consistent in rats and in mice. Simple templates were made by xerography on transparency film. Templates were used to accurately excise the barrel cortex of SmI from cortex or to place in vitro injections of HRP. The approach could be generalized to locate other cortical areas from lissencephalic brains.

Some Anatomical Bases of Cortical Somatotopic Organization; pp. 325–347

The evoked potential method was used to define the cortical visual, auditory and somatosensory areas in the mouse. Two somatotopic representations (SmI and SmII) were found which in general conform to