Somatosensory cortex: structural alterations following early injury to sense organs.

In mouse somatosensory cortex there are discrete cytoarchitectonic units, called "barrels." Each barrel is related to one sensory vibrissa on the muzzle. Individual vibrissae were carefully injured at birth; 12 to 43 days later, the corresponding barrels proved to be absent. Evidently the sensory periphery has an important influence on the structure of the somatosensory cortex.

Collateral growth and angiogenesis around cortical stroke.

BACKGROUND AND PURPOSE: We tested the hypothesis that there are significant long-term local vascular changes after ministroke that could form a basis for functional recovery. METHODS: A 6- to 8-mm cranial window was opened over the barrel cortex, which was identified by an intrinsic optical signal during mechanical stimulation of the whiskers in anesthetized female Wistar rats. Branches of the middle cerebral artery (MCA) to this region were ligated. Fluorescein isothiocyanate (FITC) transits were recorded by videomicroscopy in each rat just before, immediately after, and 30 days after ligation. Changes in surface vessels and parenchymal perfusion were measured. In similarly prepared rats, angiogenesis was identified by 5-bromo-2-deoxyuridine labeling and immunohistochemistry for the integrin family member alpha(v)beta(3). RESULTS: The intrinsic optical signal disappeared immediately after MCA ligations. FITC injection just after ligation demonstrated 3 concentric regions: 1 region of unchanged perfusion, surrounding 1 region of reduced perfusion (the ischemic border) surrounding a central core with little observable perfusion. At 30 days, the following had taken place: (1) diameters and lengths of surface collaterals in the ischemic border had grown significantly, but no new surface vessels were detected, (2) FITC entered occluded MCA segments, (3) arteriocapillary latencies in the ischemic border were shortened compared with latencies just after ligation, and (4) small infarcts were virtually identical to the poorly perfused core. Angiogenesis was confined to the ischemic border. CONCLUSIONS: Arteriolar collateral growth and new capillaries support restored perfusion in the ischemic border after ministroke and could support long-term functional recovery.

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 isothiocyanatedextrans, saline, or 3 microns 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.

Development and remodeling of cerebral blood vessels and their flow in postnatal mice observed with in vivo videomicroscopy.

Changes of blood vessels in the mouse somatosensory (barrel) cortex were assessed from birth (P0) to adulthood. Surface vessel anatomy and flow were observed directly with videomicroscopy through closed cranial windows and with intravascular fluorescent tracers. Histology was used to determine the internal capillary density. At birth, arterioles had numerous anastomoses with each other, pial capillaries formed a dense surface plexus, and pial venules and veins were relatively small and irregular. Morphological changes over the next 2 weeks included (a) fewer arteriolar anastomoses, (b) formation and growth of venules, (c) more uniform diameters of all types of vascular segments, (d) increase in intraparenchymal capillary length density (Lv), and (e) decreases in superficial capillary density and diameters. A simple morphological test showed that wall shear rates at arteriolar branch points were matched on average in neonates and adults. Flow characteristics in single vessels were evaluated. In arterioles of like diameters, (a) Vmax, (b) peak wall shear rates, and (c) peak flows were similar at all ages; (d) velocity was very high in occasional arteriovenous (AV) shunts in newborns; and (e) flow in arteriolar anastomoses was slow and variable. Although flow was heterogeneous in all types of vessel, the marked similarities in newborn and adult mice of average peak velocities and calculated wall shear rates in arterioles of the same size suggest that blood flow regulates in part the remodeling of blood vessels during development (Rovainen et al., 1992). The rodent barrel cortex undergoes major neuronal and vascular development, functional differentiation, and remodeling during the first weeks after birth. It provides special opportunities for testing how blood vessels grow and adapt to supply the local metabolic requirements of neural modules in the brain.

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 microns 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 microns 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 microns) 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.

Axonal trajectories between mouse somatosensory thalamus and cortex.

An in vitro brain slice preparation has been used to label fibers connecting the somatosensory thalamus and cortex of the mouse. In 400-800-micron brain slices, the pathway between the ventrobasal complex and somatosensory cortex was labeled under direct vision with horseradish peroxidase crystals (HRP), HRP-Nonidet P-40 (NP40) detergent chips, or a solution of HRP/dimethylsulfoxide. Thalamocortical and corticofugal fibers are organized into a plexiform system of bundles that appears to be fairly constant from animal to animal. Bundles of fibers projecting from the ventrobasal complex course between regularly spaced groups of thalamic neurons. Thalamocortical axons do not invariably leave the thalamus via the fiber bundle closest to the perikarya. Thus, nearest-neighbor relationships are abolished before these axons have even left the thalamus. The axon bundles traverse the thalamic reticular nucleus lateral to the complex. The axons then rotate about one another, analogous to the coiling of strands in rope about a central axis. This accounts for the well known 180 degrees rotation in the mediolateral direction between thalamic and cortical maps. Laterally, fiber bundles converge and diverge within the internal capsule so that nearest-neighbor relationships are lost. Individual thalamocortical axons do not bifurcate proximal to the subcortical white matter. After single bundles of fibers reach a point just below the subcortical white matter, their individual fibers diverge widely. Within the subcortical white matter most afferent fibers make a small dorsally concave loop prior to taking one of two possible courses: some of the fibers ascend directly into the overlying cortex usually angled towards the dorsal surface of the brain; other fibers run in the subcortical white matter for variable distances prior to ascending into cortex. Within somatosensory cortex, smooth axons branch near their terminals in layers IV and VI. Axonal terminal and branching patterns of these axons within somatosensory cortex are similar to those found in in vivo preparations. Most axons are smooth, but other axons are beaded. Some beaded axons project to layer I. Corticofugal fibers are labeled. Fibers leaving somatosensory cortex have an angle of descent opposite to the angle of ascent for afferent fibers, and are often fasciculated in the cortex and subcortical white matter. Within the subcortical white matter efferent fibers often loop in a direction opposite to that of afferent fibers. Corticofugal fibers occasionally give off a collateral corticostriatal branch within the internal capsule.(ABSTRACT TRUNCATED AT 400 WORDS)

New high-resolution 2-deoxyglucose method featuring double labeling and automated data collection.

A new approach to high-resolution 2-deoxy-D-glucose (2DG) emulsion-autoradiography which combines improved retention of 2DG labeling, staining with immunohistochemical and other specific markers, and automated data collection and analysis of local silver grain and stain densities is described. The Durham et al. (J. Neurosci. 1:519-526, '81) procedure for fixation of 2DG with periodate-lysine-paraformaldehyde (PLP, McLean and Nakane: J. Histochem. Cytochem. 22:1077-1083, '74) was adapted to increase retained label roughly tenfold. Phenobarbital anesthesia is induced 45 minutes after 2DG injection. Barbiturate anesthesia increases brain glycogen (Nelson et al.: J. Neurochem. 15:1271-1279, '68) and presumably increases the incorporation of intracellular 2DG from 2DG-6P into brain glycogen and other molecules (Nelson et al.: J. Neurochem. 43:949-956, '84; Pentreath et al.: Neuroscience 7:759-767, '82). Iodoacetate is added to cold fixative to prevent glycogen breakdown (Cammermeyer and Fenton: Histochemistry 76:339-356, '82). This high-resolution 2DG protocol is directly compatible with many other neuroanatomical techniques. We demonstrate 2DG emulsion autoradiography combined with cytochrome oxidase (CO) histochemistry, markers for axonal pathway tracing, plastic embedding for semithin sections, and immunohistochemical staining for glutamate decarboxylase (GAD). The method should be compatible with antibodies for other antigens and with other neuroanatomical stains. To collect the data directly from microscope slides, a computer-controlled microscope was integrated with image-processing software to eliminate the need for manual counting and scoring of autoradiograms. Regions of interest are scanned automatically at high resolution to map regional labeling and/or stain density. There is excellent correspondence between computer-enhanced two-dimensional maps of the data and the original autoradiograms. Automated counts for five specimens were compared to counts of labeled cells by trained observer. The correlation between the two sets of measurements is high (r = .93). Automated data collection has been generalized to measure regional stain densities on the autoradiographed sections for direct comparison with silver grain density. The method is extremely flexible, especially since new image-processing strategies can be developed in software to extract the desired information from materials labeled by other methods (e.g., HRP).(ABSTRACT TRUNCATED AT 400 WORDS)

High-resolution 2-deoxyglucose mapping of functional cortical columns in mouse barrel cortex.

Cortical columns associated with barrels in layer IV of the somatosensory cortex were characterized by high-resolution 2-deoxy-D-glucose (2DG) autoradiography in freely behaving mice. The method demonstrates a more exact match between columnar labeling and cytoarchitectonic barrel boundaries than previously reported. The pattern of cortical activation seen with stimulation of a single whisker (third whisker in the middle row of large hairs--C3) was compared with the patterns from two control conditions--normal animals with all whiskers present ("positive control")--and with all large whiskers clipped ("negative control"). Two types of measurements were made from 2DG autoradiograms of tangential cortical sections: 1) labeled cells were identified by eye and tabulated with a computer, and 2) grain densities were obtained automatically with a computer-controlled microscope and image processor. We studied the fine-grained patterns of 2DG labeling in a nine-barrel grid with the C3 barrel in the center. From the analysis we draw five major conclusions. 1. Approximately 30-40% of the total number of neurons in the C3 barrel column are activated when only the C3 whisker is stimulated. This is about twice the number of neurons labeled in the C3 column when all whiskers are stimulated and about ten times the number of neurons labeled when all large whiskers are clipped. 2. There is evidence for a vertical functional organization within a barrel-related whisker column which has smaller dimensions in the tangential direction than a barrel. There are densely labeled patches within a barrel which are unique to an individual cortex. The same patchy pattern is found in the appropriate regions of sections above and below the barrels through the full thickness of the cortex. This functional arrangement could be considered to be a "minicolumn" or more likely a group of "minicolumns" (Mountcastle: In G.M. Edelman and U.B. Mountcastle (eds): The Material Brain: Cortical Organization and the Group-Selective Theory of Higher Brain Function. Cambridge: MIT Press, '78). 3. Within the stereotyped geometry of the barrel field, there is considerable individual variation in the radial labeling distribution in corresponding (homotypical) columns of different cerebral hemispheres. This result is consistent with the hypothesis that dynamic processes operate to determine the connection strengths between neural elements in somatosensory cortex. It provides a basis for testing various "connectionist" and "group selection" theories of neural organization and development.(ABSTRACT TRUNCATED AT 400 WORDS)

Dendritic plasticity in mouse barrel cortex following postnatal vibrissa follicle damage.

Neonatal damage to a row of mystacial vibrissae in the mouse causes cytoarchitectonic alterations in the contralateral SmI barrel Cortex. The region for the appropriate row of barrels develops as a smaller homogeneous zone while barrels in adjacent rows are expanded. To investigate the effects of this phenomenon on the morphology of individual neurons, adult mice in which Row-C vibrissae (the middle row) had been cauterized on days 1--5 following birth were processes by the Golgi-Cox method. All neurons in layer IV of the Row-C zones, of the Row-C barrels of a control hemisphere, and some neurons in the adjacent enlarged Row-B barrels were measured with a computer-assisted microscope. Their location with respect to cytoarchitectonic boundaries was determined from a Nissl counterstain. Data from 239 cells are presented. For each cell, measures of dendritic length and branching were obtained. The orientation of the dendritic trees with respect to the barrel sides was also measured. The measures of dendritic lengths and branching did not show any differences between control and experimental animals or between animals damaged on different days. Measures of orientation did show changes related to the age at the time of damage. In animals damaged on postnatal day (PND)-3 or earlier, many cells in the Row-C zone were observed with dendrites orienting toward the adjacent Rows-B or -D. "Putative" Row-C cells in the expanded parts of Rows-B and -D were strongly oriented toward barrels in those rows. These results suggest that dendritic length and branching may be determined intrinsically but that the orientation of the dendritic trees appears to be strongly influenced by the pattern of extrinsic afferent inputs from the thalamus. In the case of the whisker-damaged animals, the orientation of the Row-C neuron dendritic trees toward the "functional" thalamocortical inputs in Rows-B and -D contributes strongly to the resultant cytoarchitectonic changes. The implications of these results for normal developmental processes and their relationship to functional studies of the cortex are considered.

Computer-assisted analyses of barrel neuron axons and their putative synaptic contacts.

The "barrels" in layer IV of rodent SmI neocortex receive inputs from individual whiskers on the contralateral face. Previous analyses of neuronal morphology in mouse and rat barrel cortex, as revealed by Golgi impregnations, have focused on the dendritic patterns of the stellate cells. The cells can be classified into two groups: Class I cells with spiny dendrites and Class II cells with smooth, beaded dendrites. These classes can be subdivided further according to somal position and spatial distribution of dendrites with respect to barrel cytoarchitectonic boundaries. In the present study the axons of these cells were examined and the locations of close appositions to dendrites of other impregnated neurons were mapped. All data are taken from Golgi-Cox preparations, cut parallel to layer IV at 140 microns, counterstained with Nissl to reveal the barrels, and measured with a computer-microscope. Axons which had extensive branching within the section (present on 10% of all impregnated cells) were chosen for measurement. The analysis of the axons revealed: (1) Class I axons are thin and directed to the white matter with recurrent collaterals in the barrels, while Class II axons are thick, frequency beaded, and directed toward the pia before cascading down into the barrels; (2) in layer IV, the axons of both cell classes tend to be as restricted to a barrel as the dendrites of the same cell are (i.e., most axons are confined to one barrel); (3) within layer IV, the Class II cell axons have a total length about three times that of Class I cell axons, and about four times as many branch points. The analysis of the appositions of these axons to impregnated dendrites of other cells revealed: (1) A majority of "contacts" tended to be made by terminal branches of the axonal trees. (2) For the Class I neurons, a greater number of appositions occur near the distal ends of complete dendritic segments. As measured from the "contacted" cell soma, appositions are more or less uniformly distributed along dendritic trees. (3) No striking patterns are found, such as an obvious propensity for axons of one cell type to prefer or avoid another cell type. These results show that the axons of barrel cells of each class are as consistent and distinctive as their dendritic trees. Specifically, the cells in each class can be distinguished by their axonal patterns on purely numerical bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Areal changes in mouse cortical barrels following vibrissal damage at different postnatal ages.

The normal cytoarchitectonic pattern of barrels in layer IV of mouse SmI face cortex is altered by early damage to the mystacial vibrissae (Van der Loos and Woolsey, '73). In the present study, the middle row of vibrissae (row-C) on one side of the face in groups of Swiss mice was cauterized on the day of birth (postnatal day [PND] -1)or on PND's - 2, 3, 4, 5, 7, 10, 12, 15, 20 and 30; littermates in each group served as controls. All animals were perfused on PND-60 and the brains sectioned parallel to SmI layer IV. For each specimen, the posteromedial barrel subfields (PMBSF) of the two hemispheres were reconstructed with a camera lucida and the cross-sectional areas of individual barrels measured using a small computer. The findings are: (1) The hemispheres ipsilateral to the vibrissal damage are quantitatively indistinguishable from the littermate controls indicating that the ipsilateral hemispheres in lesioned animals can serve as controls for observations of the type reported in this paper. (2) There are no consistent differences in the cross-sectional areas of the PMBSF's as a whole in the hemispheres ipsi- and contralateral to the peripheral damage, suggesting that there is no net loss of cortex as a result of the lesions. (3) The contralateral row-C barrels are reduced in size. Expressed as a percentage of normal values; row-C is reduced to 17% for animals lesioned on PND-1, 16% on PND-2, 38% on PND-3, 52% on PND-4 and 79% on PND-5; on PND-7 and later the cross-sectional areas of row-C barrels are normal. This implies that the barrel field of the SmI face cortex becomes progressively refractory to the effects of peripheral damage during the first postnatal week and in the period prior to PND-6, an intact periphery is necessary for normal cortical development. (4) In every case, the decreased cross-sectional area of row-C is accompanied by precisely increased cross-sectional areas of the barrels in adjacent rows-B and D. in the case of the restricted peripheral damage which we produced, there is a "compensation" in the contralateral hemisphere, which can be correlated with patterns of the specific thalamocortical projections.

The number, size and spatial distribution of neurons in lamina IV of the mouse SmI neocortex.

We located the corresponding barrel in Layer IV of the mouse SmI cortex in eleven cerebral hemispheres sectioned in a plane tangential to the pia overlying SmI and in one sectioned and prepared by a combined Golgi-Nissl method. In the section in which barrel C-1 could be optimally visualized each neuronal soma was outlined with a camera lucida and the cross-sectional area measured with the aid of a small computer. In all, nearly 7,000 neurons were measured. We estimate that on average barrel C-1 contains about 2,000 neurons. The mean cross-sectional area of the perikarya is 62.51 mu2 (S.D. plus or minus 14.51 mu2) and the size distribution of the neurons is unimodal and positively skewed. There is no segregation of cells within the barrel on the basis of size. The spatial distribution of cells in the barrel is fairly constant, from specimen to specimen, and the charactieristic cytoarchitectonic appearance of the barrel can be related to regional neuronal packing density since there are at least 1.6 as many neurons in the sides of the barrel as the hollow. The constancy of the cellular composition of the barrels indicates that the mechanisms responsible for the development of the mouse SmI cortex are fairly rigidly determined, and that the barrel field should lend itself well to further quantitative, developmental and physiological analysis.

On the "selectivity" of the Golgi-Cox method.

The randomness of the impregnation of layer IV cortical neurons by the Golgi-Cox method (Van der Loos, '56) has been assessed directly in Barrel C-1 of the mouse SmI. All Golgi-Cox impregnated neurons and unimpregnated neurons which were revealed with a Nissl counterstain were counted and measured in ten cerebral hemispheres cut tangential to the pia overlying the barrel field. The percentage of Golgi stained neurons varied considerably in different preparations from 0.73% to 2.26% with an average of 1.29%. The size distributions of both the Golgi impregnated and Nissl stained cells are similar but the difference of the means is statistically significant. However, if the means are equated there is no statistical difference in the two populations. When the Golgi precipitate is removed and the cells re-measured following Nissl staining there is a systematic reduction of the perikaryal cross-sectional area which is compatible with the differences in the means observed for the two populations as a whole. Finally, the frequency with which Golgi impregnated neurons are found in the barrel sides and hollows parallels the frequency with which Nissl stained neurons are observed in these two locations. We conclude that this variant of the Golgi method impregnates barrel neurons randomly. The value of this information for quantitative studies of cerebral cortex is discussed as is the potential of the system for elucidating some of the mechanisms responsible for Golgi impregnation.

Functional organization in cortical barrels of normal and vibrissae-damaged mice: a (3H) 2-deoxyglucose study.

The large mystacial vibrissae on the faces of rodents have punctate representations in all stations in the central trigeminal pathway, including layer IV of the somatosensory cortex (SmI). The cortical whisker correlates, multicellular units termed barrels, are not present at birth, and damage to the vibrissae during the first postnatal week results in altered adult cytoarchitectonics. The anatomical effects of vibrissae damage in the cortex have been well documented; here, we investigated the functional organization of altered SmI barrels with a high-resolution 2-deoxyglucose (2-DG) technique (Durham et al., '81, J. Neurosci. 1:519). The middle row of vibrissae was cauterized in 1-, 2-, 3-, 4-, or 5-day-old mice, and the animals were allowed to survive to sexual maturity. Various combinations of vibrissae were clipped acutely 24 hours prior to injection of 2-4 mCi of (3H)2-DG. Mice actively explored an empty cage for 60 minutes, stimulating the remaining vibrissae. The mice then were perfused and their brains prepared for paraffin histology and emulsion autoradiography. In tangential sections through layer IV, patterns of neuropil and cell body labeling were analyzed with respect to barrel cytoarchitecture in normal and vibrissae-damaged mice. In both control and experimental animals, patterns of neuropil and cell somata label corresponded exactly to barrel boundaries, whether normal or altered by vibrissae damage. Only those barrels for which vibrissae were intact had high levels of label, with anterior barrels more heavily labeled. Many neurons in the septa between these barrels and the adjacent barrels were labeled also. We found slightly higher neuropil label in the cortical zone corresponding to the damaged zone on the face in animals lesioned at any time. These data indicate that physiological somatotopy in vibrissae-damaged animals matches the anatomical cytoarchitecture.

Effects of neonatal whisker lesions on mouse central trigeminal pathways.

The mystacial vibrissae or whiskers on the face have a large representation in the rodent central nervous system. In rats and mice the projections arising from each vibrissa can be demonstrated histologically in five separate parts of the central trigeminal pathway. At every location, the pattern of the projections is isomorphic to the pattern of the facial vibrissae. For example, in the somatosensory cortex (SmI), multicellular cytoarchitectonic units in layer IV--termed barrels--correspond anatomically and functionally to the contralateral whiskers. The cortical barrels are absent at birth and their cytoarchitectonic pattern can be altered by neonatal whisker lesions. The effect is graded such that whisker damage on or after postnatal day (PND) 6 does not produce changes in the anatomical somatotopy. We undertook the present study to determine whether similar "critical periods" for susceptibility to vibrissa damage exist in the subcortical trigeminal stations of mice. In particular, we wished to find out whether subcortical projections are susceptible to whisker damage in a sequence which parallels other described developmental sequences, as has been concluded from previous work on the mouse (Woolsey et al., '79), or whether the "critical periods" are related to other aspects of development as has been concluded from work on the rat (Belford and Killackey, '80). Neonatal Swiss Webster mice sustained lesions of a single row of whiskers on PND 1, 2, 3, 4, or 5. The animals survived to adulthood. Their brains were sectioned and stained for the mitochondrial enzyme, succinic dehydrogenase (SDH), which demonstrates whisker somatotopy in all central nervous system (CNS)stations. The whisker representations at each level of the pathway, often in the same individual, were reconstructed from serial sections to assess qualitative and quantitative changes in somatotopy. Histological sections through the faces of the experimental animals were used to determine the extent of whisker damage and to show that few nerve fibers innervate the damaged zone on the face. In the brainstem representations, the zones corresponding to the damaged whiskers are shrunken and pale, regardless of the animal's age at vibrissa damage; this probably reflects the degeneration of the primary afferents. In the thalamus and the cortex, whisker damage at later postnatal times has progressively less effect on the anatomical projections patterns. Based on the changes in the projection patterns related to the damaged vibrissae and the changes in the projection patterns related to the remaining, intact vibrissae,(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology of Golgi-Cox-impregnated barrel neurons in rat SmI cortex.

Golgi-Cox-impregnated neurons in the barrel cortex of the rat were studied qualitatively and quantitatively. Adult rat brains were sectioned perpendicular to or parallel to the cortical representation of the large facial vibrissae at 125 micron. Cortical laminar and barrel boundaries were identified from the Nissl counterstain. Over 200 well-impregnated neurons in cortical layers I-IV were selected for classification and further detailed study. Three broad classes of neurons were recognized: (1) pyramidal cells with conical somata, a stout apical dendrite, and spines; (2) class I nonpyramidal cells having small spherical somata and spiny dendrites; and (3) class II nonpyramidal cells having larger ellipsoid somata and smooth or beaded dendrites. The class I cells were further subdivided into "star pyramids" (cells with an apical dendrite) and spiny stellate cells (cells in which all dendrites were of similar length). The class II cells also were subdivided into multiform cells (with multiple dendrites radiating from the soma) and bipolar cells (with two principal dendritic trunks arising from the superficial and deep aspects of the soma). The position of these various cell types in the superficial cortical laminae was mapped in sections normal to the pia. Numerous examples of the class I and class II neurons were drawn with respect to the barrels in layer IV and the extent of their processes noted. Finally, approximately 250 barrel-related class I and II neurons were studied quantitatively using a computer-microscope and digitizing tablet. The density of the Golgi-impregnated neurons corresponds to the pattern of cell density seen with the Nissl counterstain. The various cell types are not uniformly distributed as a function of cortical depth. Cells with apical dendrites were found principally in the supragranular layers and star pyramids in the superficial one-half of layer IV. Spiny stellate cells are concentrated in layer IV and the smooth cells are present in greatest number in deep layer III and deeper layer IV. On the basis of these distributions we suggest that layer IV be subdivided into two sublaminae. The class I and class II neurons can be distinguished according to quantitative criteria which apply in either plane of section used. Class I neurons have smaller projected somal areas, more proximal dendritic branching, and shorter dendrites when class I and II neurons are measured in three dimensions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute whisker removal reduces neuronal activity in barrels of mouse SmL cortex.

The autoradiographic 2-deoxy-D glucose (2-DG) method has been used to map relative changes in metabolic activity in the CNS during various functional states (Plum et al., '76). Here we describe the application of the 2-DG method to assay regional activity in the posteromedial barrel subfield (PMBSF) region of the mouse SmI cortex after acute removal of mystacial vibrissae. One day prior to isotope injection, various combinations of vibrissae (e.g., all vibrissae, row-C only, rows-B and -D only) were plucked from adult male Swiss Webster mice under anesthetic. The next day, 5 muCi of 14C-2-DG were injected into a tail vein, and the mice were allowed to actively explore an empty cage for 45 minutes. The animals were then sacrificed, the brains quickly removed, frozen, and sectioned either parallel or perpendicular to the pia at 80 mum in a cryostat. The sections were mounted, dried on coverslips, and were used to expose X-ray film, after which the sections were stained with thionin and the X-ray film developed. The tissue sections and matching autoradiograms were compared directly from photomicrographs of each. The autoradiograms showed areas of higher activity in barrels for which corresponding vibrissae were present and lower activity in barrels for which appropriate vibrissae were missing. In tangential sections from animals with all vibrissae intact, the PMBSF was uniformly and consistently higher in activity than in cases with all vibrissae missing. The removal of row-C or rows-B and -D resulted in strips of decreased activity in the corresponding PMBSF rows. The same patterns of increased or decreased activity were also seen in sections normal to the pia, but the changes in activity, while greatest in layer IV, extended through all layers of the cortex. Finally, in a number of the autoradiograms, density patterns could be recognized which later were shown to relate directly to sides of individual barrels. The results indicate: (1) Acute removal of the peripheral vibrissal hairs is sufficient to deprive the related contralateral cortical barrel neurons of normal activity. Thus in the mouse somatosensory system it may be possible to determine the relative importance of sensory deprivation and neonatal peripheral lesions in normal cortical development. (2) The barrels are part of a functional cortical columnar organization similar to that in other sensory systems. And, (3) the 14C-2-DG-X-ray technique is sufficiently sensitive to reveal parts of individual barrels in autoradiograms and thus, with some modification, may be suitable for the study of small populations of neurons.

Comparative anatomical studies of the SmL face cortex with special reference to the occurrence of "barrels" in layer IV.

In the SmL cortex of mice and rats there are cytoarchitestonically identificable groups of cells -- called barrels -- some of which have been shown to be directly related to whiskers and other sensory hairs on the contralateral face. In this study we have used a comparative approach to determine the incidence and variation of the barrels. The brains of 27 mammalian species have been examined histologically to determine whether barrels exist in layer IV of what is known or likely to be the face area of SmI. Thick sections (50-100 mum) were taken tangential to the pia overlying SmI and stained with thionin. The patterns of facial whiskers were also mapped by dissection of the facial skin. Barrels were seen only in brains of species belonging to three of the seven mammalian orders examined. We have confirmed Weller's ('72) observation of barrels in the Australian brush-tailed possum but have not found barrels in two marsupials from the western hemisphere. Barrels were demonstrable in representatives of four of five rodent suborders examined and in the rabbit. From the study of the rodent brains, a number of trends emerge. (1) The organization of the barrel fields is "dictated" by the organization of the sensory periphery. Animals with five rows of large mystacial (moustache-like) vibrissae have five rows of PMBSF (Posteromedial barrel sub-field) barrels. (2) The barrels are confined to layer IV of (what is known or likely to be) the SmI face area. The pattern and cortical location of the barrel field is consistent among different specimens of the same species. (3) Certain behavioral patterns do not preclude the existence of the barrels. Species which possess well developed visual systems and behaviors (e.g., grey squirrel) and forms which do not actively explore the environment by whisking their vibrissae (e.g., guinea pig) have barrels. (4) Within a given rodent suborder, the barrels become more difficult to identify, as the brains become larger. We have not yet been able to demonstrate barrels in the largest rodent, the capybara.

2-DG uptake patterns related to single vibrissae during exploratory behaviors in the hamster trigeminal system.

Stimulation of one or several whiskers activates discrete foci throughout the trigeminal (V) neuraxis. These foci contribute to patterns, corresponding to the patterns of vibrissae, that have been directly related to aggregates of cells and axon terminals in the "barrel" cortex. Here, we combine high-resolution, 2-deoxyglucose (2DG) mapping and cytochrome oxidase (CO) staining to determine whether the known pattern of V primary afferent projections is sufficient to deduce the functional activation of their targets during exploratory behavior. Four adult hamsters had all of their large mystacial vibrissae trimmed acutely, except for C3 on the left, and B2 and D4 on the right; in two others, the left C3 and right A1 and E4 whiskers were spared. After fasting overnight, 2DG was injected and the animals behaved freely in the dark for 45 minutes. The brainstem, thalamus, and cortices were sectioned, then processed for both CO staining and 2DG autoradiography. Image-processing microscopy was used to separate the autoradiographic silver grains from the histochemical staining. CO patches were patterned in a whisker-like fashion in the full rostrocaudal extent of V nucleus principalis and in caudal portions of spinal V subnuclei interpolaris and caudalis, but absent in subnucleus oralis. 2DG silver grains were densest above those CO patches in the pattern corresponding to the active whiskers. There were no consistent 2DG foci in subnuclei oralis or rostral caudalis. In these same cases, prominent 2DG labeling was restricted to the appropriate barrels in the contralateral cortex. Only one case, however, displayed a clear and appropriate region of heightened 2DG uptake in contralateral ventroposteromedial thalamus (VPM) and the adjacent part of the reticular thalamic nucleus. Patterns of increased glucose utilization with single whisker stimulation are well matched to the CO patterns that mirror distributions of neurons associated with a vibrissa in the V brainstem complex, thalamus, and cortex. Single whiskers are represented by relatively homogeneous longitudinal columns of 2DG labeling in the V brainstem nuclei. The columns are not continuous through the axial extent of the V brainstem complex; rather, they occur separately within principalis, interpolaris, and caudalis. While whisker columns were consistently labeled in interpolaris and caudalis in all animals, the labeling was increasingly variable in principalis, barrel cortex, and VPM, respectively. This suggests that the behaving animal can and does significantly modulate activity in this major, synaptically secure pathway.

Local intra- and interlaminar connections in mouse barrel cortex.

Focal injections of horseradish peroxidase (HRP) in dimethylsulfoxide (DMSO) were targeted into mouse somatosensory cortex, in vitro, with a template. Injections were made at different depths and in different locations in the whisker-barrel-defined somatosensory map in order to determine quantitative connectivity patterns within and between barrel-defined cortical columns. Cortices were sectioned in a plane parallel to the pia at 75 microns. Data were collected directly from microscope slides by computer. Data are presented as: 1) Plots of computer-mapped HRP reaction product density in neurons and cell locations for each section in relation to barrel boundaries; 2) histograms of label in cortical layers related to individual barrel-defined columns; 3) polar plots of relative amounts of label within individual barrel columns in sections through each barrel column; 4) vectors which represent HRP reaction product density as a function of direction and distance from the injection site; 5) statistical analysis of the shape of the label distribution pattern in the plane of the cortex as a function of injection site depth; and 6) probability of labeling of any other barrel column given a labeled barrel column. The principal findings are: 1) The pattern of label distribution, after an injection directly above or directly below an individual barrel, is hour-glass shaped with the waist of the hour-glass in layer IV. 2) Connections within barrel cortex are asymmetrical. Barrel-related columns within a row are more strongly interconnected than those in different rows. 3) Connections of the small barrels associated with whiskers on the upper lip are strongest with other small barrels, but strong connections also exist between these small barrels and the larger barrels. 4) The pattern of intracortical connections in SII is not asymmetrical; interlaminar connections in SII are fundamentally different from those in barrel cortex. 5) Quantitative intracortical projection patterns are highly consistent with functional data on intracortical processing of whisker information. As such, the quantitative data clearly indicate the spatial extent and relative magnitude of populations of neurons involved in intracortical processing of sensory information. The spatial arrangements of these intracortical connections, in conjunction with known developmental events, make it highly likely that the distribution of intracortical axons in mouse barrel cortex is sculpted in part by experience.