The "neostriatum" develops as part of the lateral pallium in birds.

Telencephalic organization in birds is so unusual that many homologies between avian and mammalian telencephalic areas remain controversial. Particularly contested is the avian "neostriatum," which has historically been homologized to either mammalian striatum, lateral neocortex, or endopiriform claustrum. Because homologies between these adult structures have been so difficult to resolve, we have begun to examine how telencephalic development diverges between birds and other vertebrates. To this end, biotinylated dextran was injected into the lateral telencephalon of chick embryos at 3 d of incubation, and the distribution of labeled cells was examined up to 14 d later. The data show that a definite boundary to cellular migration develops just ventral to the neostriatum between 5 and 8 d of incubation. Labeled polyclones within the neostriatum stretch from the ventricular zone to the brain surface and exhibit an increasingly rostrocaudal orientation as development proceeds. Individual polyclones contribute cells to several of the distinct auditory, visual, somatosensory, and olfactory regions within the neostriatum. A comparative analysis suggests that the avian neostriatum develops from a precursor region that in other vertebrates gives rise to olfactory cortex and, when present, to other components of the piriform lobe, such as the endopiriform claustrum and basolateral amygdala. Conclusions about lateral pallial homologies between birds and mammals remain uncertain, however, primarily because so little is known about the development of the lateral pallium in mammals. This lacuna might be filled by applying to mammals the novel fate-mapping method described in the present paper.

Distribution of radial glia in the developing telencephalon of chicks.

Radial glia are known to have a sparse and uneven distribution in the telencephalon of adult birds. The present study utilizes antibodies against vimentin to reveal a more extensive, and more clearly radial, set of radial glia in the chicken telencephalon during the first half of embryogenesis. This initially extensive radial glial fiber system becomes distorted and reduced between 10 and 14 days of incubation. This reduction coincides with the cytoarchitectural differentiation of the telencephalon into its major adult subdivisions. Because developing neurons tend to migrate along radial glial fibers in both birds and mammals, a topological projection of these major subdivisions onto the embryonic ventricular zone along the radial glial fibers suggests hypotheses about lineage relationships that can be tested by subsequent experimental methods. This analysis suggests that the major components of the avian dorsal ventricular ridge, i.e., the ventral hyperstriatum, the neostriatum with its various subdivisions, part of the archistriatum, and probably also the piriform cortex, all derive from overlapping portions of the lateral pallial ventricular zone. Staining with antibodies against neurofilament suggests that this developmental parcellation of the lateral pallial complex is associated with the development of neuronal fiber systems.

Myocardial sarcolemma in ischemia: a quantitative freeze-fracture study.

Freeze-fracture electron microscopy permits the visualization of the intramembrane particles (IMP). These IMPs are presumably proteins responsible for the main functions of the membrane. Quantitative techniques (Clark-Evan statistics) were applied to determine in a critical manner whether IMP pattern shifts (random, clustered, or ordered) occur under the ischemic conditions (5-45 min with and without reperfusion) and whether this change is related to the experimental condition. In each case three hearts, eight replicas/heart, one area of 0.25 micron 2 of membrane fracture face/replica was measured to give a total of 6 micron 2 of membrane counted for each condition (control vs. ischemic). A mixed effects nested model analysis of variance was performed in each variable. We found that IMP aggregation can be present in some control membranes, but the degree of aggregation was greater and more consistent in membranes made ischemic and followed by reperfusion. Most striking was the significant clustering of IMPs in membranes from hearts ischemic for only 5 min. Reperfusion after only 5 min of ischemia reversed IMP clustering. Functionally at this time there is an increase in K+ concentration in the interstitial space that reaches approximately 15 mM within 10 min and reverses on reperfusion. The structural alteration in IMPs appears to parallel the function in ischemic hearts.

Membrane structure in ultrarapidly frozen, unpretreated, freeze-fractured myocardium.

Ultrarapid freezing has been applied to monitor the structure of the freeze-fractured myocardial sarcolemma. Our two goals were to demonstrate that large areas of membrane can be preserved free of visible ice crystal damage and, thus, be amenable to quantitative analysis and to compare the structure of directly frozen myocardial membranes with conventionally prepared tissue. The E face was most affected by lack of chemical pretreatment. First, our laboratory reported an increase in E face particle density from 379 +/- 30/micron 2 in conventional fixed tissue to 489 +/- 18/micron 2 in unpretreated tissue. Discrete arrays of 12-15 nm particles on the E face were a striking feature of the unfixed sarcolemma. However, P face intramembrane particle (IMP) density remained unchanged from previous estimates in fixed tissue. Specialized regions of the sarcolemma were enhanced in ultrarapidly frozen tissue. Particle domains of the adherens junctions were very prominent in forming a cap alongside the gap junctions. Both the P and E faces of the gap junctions were highly ordered into hexagonal arrays. Caveolae in the membrane were infrequent in both P and E faces.

Intercellular connections in rabbit heart as revealed by quick-frozen, deep-etched, and rotary-replicated papillary muscle.

Rabbit papillary muscles were ultrarapidly frozen, fractured, deep-etched, and rotary shadowed. These techniques revealed the interstitial space where the complex network of fine microthreads that connect myocytes to each other and to collagen fibrils can be seen in a three-dimensional array similar to scanning electron micrographs but at a resolution attainable in freeze-fracture microscopy.

Ultrastructure of isolated sarcolemma from dog and rabbit myocardium. Comparison to intact tissue.

We compared the morphology of cardiac sarcolemmal membranes isolated from dog and rabbit hearts with the sarcolemma in intact cells, using freeze-fracture and thin-section electron microscopy. In addition, we estimated the sidedness of the isolated sarcolemma based on its freeze-fracture morphology and biochemical determinations of sialic acid content and Na,K-ATPase activity. The bilayer in isolated membranes is similar, in its morphology, to intact membrane. The isolated sarcolemmal vesicles have the same density of intramembrane particles per micron2 as the intact sarcolemma (approximately 2800/micron2). The particle counts in isolated sarcolemma were very homogeneous, with the peak in particle density curve the same as found for intact cells. In the intact myocardium, both sarcolemmal and transverse tubular membranes have the same density of intramembrane particles. Thin-section morphology of the isolated sarcolemma shows an intact surface coat, whereas portions of the external lamina are absent. Both the biochemical and morphological data indicate that there is a substantial fraction of inside-out and right side-out vesicles in this preparation. Considering the approximations inherent in both morphological and biochemical approaches, we find the qualitative agreement of the estimations of vesicle orientation noteworthy.

Calcium depletion in rabbit myocardium. Ultrastructure of the sarcolemma and correlation with the calcium paradox.

The ultrastructure of the Ca-depleted myocardial sarcolemma (via Ca-free and Ca-free plus EGTA perfusion at 28 degree C and 37 degree C) was studied in the vascularly perfused interventricular septum of the rabbit. Thin-section and freeze-fracture electron microscopy was used. Two major structural defects in the sarcolemma were found. (1) Ninety percent of the Ca-depleted cells have between 30 and 40% of their glycocalyx separated from the bilayer. With tannic acid staining, the separation is seen to occur between the external lamina and the surface coat. (2) Freeze-fracture data showed an apparent decrease in intramembrane particles on the P face of unidirectionally shadowed replicas. Quantitation of rotary-shadowed replicas showed no decrease in density of intramembrane particles. It was concluded from this that there was no loss of intramembrane particles, but rather a reorientation in the plane of the bilayer after Ca depletion. Both glycocalyx and bilayer changes were present after perfusion of the heart for only 5 minutes (37 degree C) with Ca-free perfusate. With low temperature and Cd substitution, separation of the glycocalyx occurred in less than 1% of the cells. After Ca depletion at 18 degree C, the density of intramembrane particles on the P face was not significantly different from controls. Cd substitution did not prevent the decrease total intramembrane particles per square micron, but the larger intramembrane particles had similar densities (154/micrometer2) as control (181/micrometer2), and as Ca-depletion with hypothermia (180/micrometer2). These findings indicate that structural changes in the glycocalyx and the bilayer can be totally prevented by hypothermia. Cd, on the other hand, prevents glycocalyx separation and affords protection only to the large intramembrane particles. Upon reperfusion with Ca, the intramembrane particles undergo the further alteration of aggregation, while numerous vesicles can be seen in the fracture plane of the membrane.

Structure of the freeze-fractured sarcolemma in the normal and anoxic rabbit myocardium.

The purpose of this study was to examine the ultrastructure of the sarcolemma in the normal and severely anoxic rabbit heart with the technique of freeze-fracture. Severe anoxia and subsequent reoxygenation cause a significant decrease (31%) in intramembranous particles (IMP) in the P face of the membrane and a 25% decrease in the E face. P face IMP's are severely aggregated. The decrease in density and the redistribution of IMP's indicate a severely altered lipoprotein structure of the sarcolemma. In addition, the necks of caveolae open and the caveolae become flattened in the plane of the membrane. With reoxygenation, many rupture. Spherical projections of cytoplasmic vesicles appear in the membrane (possibly of sarcoplasmic reticulum or lysosomal origin) and also can be seen to rupture after reoxygenation. When glucose is present in the perfusate, it affords some protection against these structural defects. We propose that the fragmentation or holes in the sarcolemma reported in severe anoxia are directly related to the structural changes reported in this study.