Restricted neuronal expression of ubiquitous mitochondrial creatine kinase: changing patterns in development and with increased activity.

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II-III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.

New patterns of intracortical projections after focal cortical stroke.

Cortical strokes alter functional maps but associated changes in connections have not been documented. The neuroanatomical tracer biotinylated dextran amine (BDA) was injected into cortex bordering infarcts 3 weeks after focal strokes in rat whisker barrel (somatosensory) cortex. The mirror locus in the opposite hemisphere was injected as a control. After 1 week of survival, brains were processed for cytochrome oxidase (CO)-, Nissl-, and BDA-labeled neurons. Cortex bordering the infarct (peri-infarct cortex) had abnormal CO and Nissl structure. BDA-labeled neurons were plotted and projections were analyzed quantitatively. Animals with small strokes had intracortical projections, arising from peri-infarct cortex, not seen in normal hemispheres: the overall orientation was statistically significantly different from and rotated 157 degrees relative to the controls. Compared to the controls, significantly fewer cells were labeled in the thalamus. Thus, after focal cortical stroke, the peri-infarct cortex is structurally abnormal, loses thalamic connections, and develops new horizontal cortical connections by axonal sprouting.

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.

Postnatal growth of intrinsic connections in mouse barrel cortex.

Surprisingly little is known about the development of connections within a functional area of the cerebral cortex. We examined the postnatal growth of connections in mouse barrel cortex during the second and third weeks after birth, coinciding with the period of rapid synaptogenesis that occurs just after the barrels first form. A barrel is a group of neurons in layer 4 of somatosensory cortex that is part of a cortical column. Each whisker/barrel column is linked anatomically and functionally to a homotopic whisker on the contralateral face. Radial groups of cortical neurons were labeled with the neuronal tracer biotinylated dextran amine in mice ranging in age from postnatal day 8 (P8; P0 is the date of birth) to adulthood. The spatial distributions of retrogradely labeled neurons in different laminae were analyzed. The barrel map in layer 4 was used as a template to compare quantitative data from different animals and to account for substantial changes in barrel and barrel field size during development. Intrinsic projections 1) innervate increasingly more distant targets within barrel cortex up to 3 weeks of age; 2) continue to form in targets after 3 weeks, effectively strengthening existing connections; 3) follow a timetable for growth that is layer-specific; 4) link more distant barrel columns in layer 4 from neurons that are found preferentially in the barrel side and the septa between barrels; and 5) form over the shortest distances between the barrel columns. These data indicate that intrinsic connections in mouse barrel cortex develop by the progressive addition of neuronal connections rather than by sculpting preliminary connections. We describe statistically significant changes in connectivity during development that may be applied to model and assess the development of connections after a variety of experimental perturbations, such as to the environment and/or the genome.

Gliomas in rodent whisker barrel cortex: a new tumor model.

OBJECT: Surgical treatment of gliomas is difficult because they are invasive. Invasion of essential cortex often limits or precludes surgical resection. A tumor model was developed in which the rodent whisker barrel cortex was used to examine how gliomas affect cortical function and structure. METHODS: Both DBT (mouse) and C6 (rat) glioma cell lines were grown in culture and labeled with the fluorescent marker Dil in vitro. Labeled tumor cells were then injected into the whisker barrel cortex of adult mice and rats. Neurological assessments were made daily and magnetic resonance (MR) images were obtained. Animals were killed by perfusion 6 to 14 days after injection, and histological sections were prepared and studied. Tumors were found in all 20 rats and 10 mice that had been injected with the C6 and DBT cell lines, respectively. The animal cells had been labeled with Dil in vitro, and all in vivo tumors proved to be Dil positive. The MR images revealed the tumor locations and serial MR images demonstrated tumor growth. Histological evaluation confirmed the location of the tumor and the disruption of barrel cortex architecture. CONCLUSIONS: Both DBT and C6 glioma cell lines can be used to generate malignant glial tumors reproducibly in the whisker barrel cortex. Fluorescent labeling and cytochrome oxidase staining permit visualization of tumor growth patterns, which disrupt the barrel cortex by microscopic invasion and by gross tissue deformation. Magnetic resonance imaging demonstrates the anatomical extension of these tumors in live rodents. Using this model for further studies on the effects of malignant glioma growth on functional cerebral cortex should advance our understanding of the neurological issues and management of patients with these tumors.

Increased brain capillaries in chronic hypoxia.

The effect of chronic hypobaric hypoxia (28 days, 455 Torr) on the organization of brain vessels was studied in Balb/c mice. In comparison to age-matched controls kept at sea level, emulsion-perfused capillaries in hypoxic mice showed marked dilation in all brain areas studied. Capillary length per unit volume of tissue (Lv) was increased in the cerebellar granular layer, the caudate nucleus, the globus pallidus, the substantia nigra, the superior colliculus, and the dentate gyrus. There was a selective increase of Lv in the hippocampus (CA1 strata pyramidale and lacunosum and CA3 strata pyramidale and oriens) and in somatosensory cortex layers V and VI, motor cortex layers II, III, V, and VI, and auditory cortex layers II and III. An increase in capillary surface area per unit volume of tissue was also determined in several brain areas, including layer IV of somatosensory cortex, where Lv was not significantly increased. The O2 diffusion conductance and PO2 in the tissues were estimated with a mathematical model. The remodeling of capillary diameter and length during chronic hypoxia accounts for the significant increase of O2 conductance to neural tissues. Also the estimated tissue PO2 in chronic brain hypoxia is markedly increased in the caudate nucleus and the substantia nigra compared with acute hypoxia. These results suggest that formation of new capillaries is an important mechanism to restore the O2 deficit in chronic brain hypoxia and that local rates of energy utilization may influence angiogenesis in different areas of the brain.

Local cerebral blood flow during the first hour following acute ligation of multiple arterioles in rat whisker barrel cortex.

The objectives are to measure the early time-course of the flows of blood, red cells, and plasma in brain tissue destined to infarct following arterial occlusion. The flux of fluorescent red blood cells (fRBCs) through venules and the arteriovenous transit times (AVTT) of fluorescein-labeled plasma albumin were periodically monitored in anesthetized adult Wistar rats before and up to 60 min after permanent ligations of several small branches of the middle cerebral artery. Of note, fRBC is a function of venular erythrocyte flow and volume, whereas AVTT is a function of plasma flow and volume in visible arteriole-capillary-venule units. In another group of anesthetized rats, local cerebral blood flow (ICBF) was measured 1 h after permanent arterial occlusion by [14C]iodoantipyrine (IAP) autoradiography. With this model of focal ischemia, the lesion is highly reproducible and involves part of the whisker barrel cortex. Infarction of this area was observed in 12 of 13 rats. From 10 to 60 min after arterial occlusion, AVTT was nearly four times longer in the ischemic barrel cortex than at the same site before ligations, and fRBC flux was 25%. Neither parameter changed appreciably over this time. After 60 min of ischemia, ICBF on the ipsilateral barrel cortex was 18% of that on the contralateral side and 15% of the sham control value for the same area of the barrel cortex. Since whole blood flow in the ischemic barrel cortex was < 20% of normal at 60 min and AVTT and fRBC flux were essentially constant from 10 to 60 min, the rates of plasma and red cell flows were similarly depressed during the first hour of arteriolar occlusion. In conclusion, such lowering of red cell, plasma, and blood flows produced consistent infarctions in the barrel cortex.

Blood flow dynamics in different layers of the somatosensory region of the cerebral cortex on the rat during mechanical stimulation of the vibrissae.

The present work reports studies of the quantitative spatial and temporal characteristics of changes in local blood flow in different layers of the somatosensory cortex of rats during adequate mechanical stimulation of the vibrissae. Studies were performed using 34 Wistar rats. Skull trepanning was performed under urethane (1 g/kg) anesthesia. Television-guided microscopy was used to introduce a set of three platinum electrodes (100 microns in diameter, with tip diameters of 30-40 microns) into the somatosensory cortex projection zone of the vibrissae. The first and third electrodes were positioned in cortical layers I-III and IV-VI and the central electrode was used to generate hydrogen within the tissue. Electrode positions were confirmed histologically after experiments. Animals were placed on artificial ventilation and one or all vibrissae were stimulated at a frequency of 3 Hz for 60 sec, with interstimulus intervals of 3 min. Changes in the local blood flow were measured during stimulation and for 1 min afterwards, using the hydrogen clearance method, and brain tissue impedance was also measured. There was a small (up to 5-7%) reduction in blood flow in the first seconds of stimulation, which was followed 15-25 sec later by an increase and subsequent return to initial when stimulation stopped. The increases in blood flow during stimulation of all vibrissae were by 24.2 +/- 6.7% (n = 36) in layers IV-VI and 24.5 +/- 5.6% (n = 34) in layers I-III; increases in response to stimulation of single vibrissae were by 19.4 +/- 7.4% (n = 28) and 17.8 +/- 6.4% (n = 28) respectively. The dynamics of impedance changes corresponded to those of blood flow changes. Thus, heterogeneity was found in changes of local brain blood flow in different layers of the somatosensory cortex during increases in cortical functional activity.

[Dynamics of local cerebral blood flow in different somatosensory cortical layers during whisker stimulation in rats]. / Dinamika krovotoka v razlichnykh sloiakh somatosensornoi oblasti kory golovnogo mozga u krys pri mekhanicheskoi stimuliatsii vibriss.

Whisker stimulation in rats was found to increase the local cerebral blood flow (lCBF), its SD and to damp slow oscillations. It was established that lCBF drops slightly within a few seconds after the stimulus onset. The data obtained suggest that lCBF evoked sensory stimulation changes are distinctly localized in different layers of the somatosensory cortex.

Activation of a wide-spread network of inhibitory neurons in barrel cortex.

The one-to-one correspondence of whiskers to barrels in layer IV of rodent somatosensory cortex can be demonstrated by a precise match between columns of heavy 2-deoxyglucose (2DG) label in layer IV barrels and other layers which correspond to stimulated whiskers. While there is specificity of peripheral-to-central mapping, the extent to which integration and/or modulation are generated by circuitry within or interactions between the barrel-defined whisker columns is not clear. Following stimulation of selected whiskers, large cells at the layer IV-V boundary throughout the barrel field are heavily labeled by 2-deoxyglucose (2DG) at high resolution. Many of these cells are outside the barrel columns of the stimulated whiskers. Further, the number of cells labeled is not directly related to the number of activated barrel columns. These neurons are not labeled in animals anesthetized before 2DG injection and are not as heavily labeled in barrel fields of somnolent animals. Most of the heavily labeled neurons immunolabel for glutamate decarboxylase (GAD) and are presumed to be inhibitory, while a smaller number of labeled neurons, presumed to be excitatory, immunolabel for glutamate (Glu). Similar populations of large, heavily 2DG-labeled neurons are found in other cortical areas. These relatively few neurons are exceptionally active and may modulate integrative functions of cerebral cortex.

The neocortical local circuit--a research workshop held in Sde-Boker, Israel, May 4-8, 1997.

This report does not capture the raw vitality of the Sde-Boker workshop. We did not anticipate that so many presentations would focus on the importance of spike timing in the operations of cortical microcircuits and the profound effect of this on other cortical and subcortical networks. The roles of individual spikes, temporal firing patterns, neuronal synchronization, intrinsic neuronal and network oscillation, coincidence detection, integration by individual cells or even individual dendritic spines were also strong themes of nearly all presentations. The cortex could be viewed as a network, which, is similar to an orchestra, where each neuron (musician) generates particular timed spike patterns (notes) for which 'every spike counts' (the symphony). This assertion would have been the butt of considerable skepticism less than a decade ago. The conference led to a view that the cerebral cortex is a spatially distributed timing network. As such, the consensus reached provides a conceptual framework for exciting new discoveries in the next decade. One of the truly exciting outcomes of the workshop was the seamless integration of concepts derived from studies employing direct approaches such as brain slices to cell cultures and computer simulations to whole animal physiology. The availability of an ever expanding armamentarium of increasingly sophisticated techniques and cross-fertilization promises develop the field in a direction that is as flexible and as subtle as the cortical tissue it seeks to explain. It was perhaps symbolic that this meeting was held at a place in the desert where David Ben-Gurion, the first Prime Minister of the modern state of Israel, had a vision of pioneers settling the untamed Negev that had intrigued and intimidated mankind for so long. The hauntingly beautiful Negev, like the cerebral cortex, was an unyielding frontier. Now thanks to modern ingenuity and technology the Negev can, like the cerebral cortex, finally be conquered.

Neuronal units linked to microvascular modules in cerebral cortex: response elements for imaging the brain.

How neuronal activity changes cerebral blood flow is of biological and practical importance. The rodent whisker-barrel system has special merits as a model for studies of changes in local cerebral blood flow (LCBF). Stimulus-evoked changes in neural firing and 'intrinsic signals' recorded through a cranial window were used to define regions of interest for repeated flow measurements. Whisker-activated changes in flow were measured with intravascular markers at the pia. LCBF changes were always prompt and localized over the appropriate barrel. Stimulus-related changes in parenchymal flow monitored continuously with H2 electrodes recorded short latency flow changes initiated in middle cortical layers. Activation that increased flow to particular barrels often led to reduced flow to adjacent cortex. Dye was injected into single penetrating arterioles from the pia of the fixed brain and injected into arterioles in slices of cortex where barrels were evident without stains. Arteriolar and venular domains at the surface were not directly related to underlying barrels. Capillary tufts in layer IV were mainly coincident with barrels. The matching between a capillary plexus (a vascular module) and a barrel (a functional neuronal unit) is a spatial organization of neurons and blood vessels that optimizes local interactions between the two. The paths of communication probably include: neurons to neurons, neurons to glia, neurons to vessels, glia to vessels, vessels to vessels and vessels to brain. Matching a functional grouping of neurons with a vascular module is an elegant means of reducing the risk of embarrassment for energy-expensive neuronal activity (ion pumping) while minimizing energy spent for delivery of the energy (cardiac output). For imaging studies this organization sets biological limits to spatial, temporal and magnitude resolution. Reduced flow to nearby inactive cortex enhances local differences.

Rapid optical imaging of whisker responses in the rat barrel cortex.

Videomicroscopy was used to image 'intrinsic' responses over the rat barrel cortex through a closed cranial window during controlled whisker stimulation. With a Macintosh IIfx running Image 1.49 VDM, video frames from a CCD camera were captured and averaged before, during and after whisker stimulation. The technique presented here is a functional imaging modality--using conventional videomicroscopic equipment, a small computer, and public domain NIH Image software--with a temporal resolution of 33 ms. Images can be obtained directly from the CCD camera or recorded to videotape for post hoc analysis. Pixel by pixel comparison of prestimulation images to images obtained during stimulation revealed changes in the reflectance characteristics of cortex and vessels overlying the barrel field. Imaged responses superimposed on barrel histology to map intrinsic signal matched barrels of the stimulated whiskers in every case. Video imaging of the rat barrel cortex provides a useful method for rapid targeting for other experimental protocols and has potential for analyzing localized responses to physiologic stimuli in vivo.

[Cerebral blood flow and cerebrovascular reactivity in the early postnatal ontogeny of rats]. / Mozgovoi krovotok i tserebrovaskuliarnaia reaktivnost' v rannem postnatal'nom ontogeneze krys.

The development of cortical cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) were studied in Wistar rats during early postnatal ontogenesis, in groups aged 2-5, 6-8, 9-11, 12-15, 16-18 and 19-25 days. CBF was measured polarographically using inhaled hydrogen clearance method, with platinum electrodes inserted into parietal cortex. At the mentioned age periods, CBF and its percentage of adult level averaged as 38 (21%), 81 (45%), 142 (78%), 85 (47%), 110 (61%), and 118 (65%) ml/100 g/min, respectively. Hence, during the early postnatal ontogenesis CBF increased gradually, however, it did not reach the adult level up to the end of the first month. CBF peak at 9-11 days period is suggested as a result of sharp rise of the brain vessels growth. CVR was assessed as the percentage of CBF increase after standardized 5% CO2 inhalation test. At the above age periods, CVR was found to bi 26, 33, 36, 41, 44 and 31% respectively, and was similar to adult rat CVR of 36%. The conclusion was drawn that regulatory mechanisms of adequate responses of brain vasculature to chemical metabolic factors are well developed already at the very beginning of postnatal life.