Glucose production by lamprey meninges.

Meningeal tissue of the lamprey larva produces glucose during inicubation and contains glucose-6-phosphatase and large amounts of glycogen. Little glucose is liberated by brains from which meninges have been removed. This primitive meningeal tissue may produce glucose for use by the brain during periods of metabolic stress.

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.

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.

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.

Regeneration of Müller and Mauthner axons after spinal transection in larval lampreys.

The regeneration of large unmyelinated axons following transection of the spinal cord was studied in small larval sea lampreys (Petromyzon marinus). Individual Müller and Mauthner axons normally occur in a characteristic pattern in the spinal cord, but their positions were altered in the first several segments caudal to the lesion following regeneration. Müller axons grew out of the ventral tracts and sometimes looped back towards the brain or crossed the midline; maximum misdirection of axons occurred near the site of transection Mauthner axons frequently bifurcated. Despite the aberrant and incomplete regeneration of axons, the larvae exhibited normal coordinated swimming ,crawling, and coiling behavior.

Counts of axons in electron microscopic sections of ventral roots in lampreys.

The number of motoneurons in the lamprey spinal cord was estimated by counting axons in electron microscopic sections of ventral roots. Physiologically identified and marked motoneurons had one axon each in an ipsilateral ventral root. The corresponding axons in sections were unmyelinated, individually ensheathed, and 2-20 micron in diameter. Lampreys lack an organized sympathetic nervous system, but a minor and variable population of small axons with dense-cored vesicles was also present in ventral and dorsal roots. The estimated numbers of motor axons per ventral root in adult Ichthyomyzon unicupis and I. castaneus were 70.6 +/- 9.3 (mean +/- SD), in adult landlocked Petromyzon marinus 100.1 +/- 15.0, and in large ammocoetes of anadromous Petromyzon marinus 106.8 +/- 14.3. The total estimated numbers of myotomal and fin motoneurons were as many as 13,000 in the spinal cord of adult Ichthymyzon and 24,000 in adult Petromyzon.

Generation of respiratory activity by the lamprey brain exposed to picrotoxin and strychnine, and weak synaptic inhibition in motoneurons.

The roles of Cl-dependent synaptic inhibition in the generation of fictive breathing were tested in isolated brains of adult lampreys, Ichthyomyzon unicuspis. Only a few inhibitory synaptic potentials were recorded in respiratory motoneurons between excitatory bursts. This was also true after Cl- injections inverted them to depolarizing potentials. A weak and variable phase of Cl-sensitive synaptic inhibition occurred at the ends of excitatory bursts. Respiratory motoneurons had a pronounced post-spike hyperpolarization, which was distinct from synaptic inhibition and appeared to be a more important mechanism for termination of firing. The production of the basic rhythm for respiration was tested in strychnine, picrotoxin, bicuculline and Cl-free fluid. Low concentrations of the blocking drugs prevented the inhibitory effects of bath-applied glycine and gamma-aminobutyric acid, but essentially normal respiratory bursts still occurred. Equilibration of isolated brains in high concentrations of strychnine and picrotoxin did not prevent periodic activities, but burst durations were increased and inter-burst intervals were longer and less regular than normal. Similar bursts could also occur transiently in Cl-free fluid. Recordings from the IX and X motor nuclei indicated that respiratory neurons produced the periodic bursts in the presence of strychnine and picrotoxin. Hemisections of the brain behind the V motor nuclei eliminated the bursts ipsilaterally. This indicated that descending excitation was necessary during pattern generation both in normal fluid and in the presence of antagonists of synaptic inhibition. Conventional synaptic inhibition does not appear to be essential for respiratory pattern generation in the adult lamprey but may contribute to its modulation. The hypothetical neural oscillator may consist of excitatory bursting interneurons.

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.

Spinal and medullary dorsal cell axons in the trigeminal nerve in lampreys.

The ophthalmic branch of the trigeminal nerve was labeled with horseradish peroxidase (HRP) in 3 species of adult lampreys. In each species, some medullary and spinal dorsal cells were retrogradely labeled by HRP. Approximately 30% of spinal dorsal cells were labeled ipsilateral to the injection; an occasional contralateral spinal dorsal cell was also labeled. Intracellular recordings confirmed the anatomical findings.

N-Methyl-D-aspartate (NMDA), kainate and quisqualate receptors and the generation of fictive locomotion in the lamprey spinal cord.

The motor pattern underlying swimming can be elicited in an in vitro preparation of the lamprey spinal cord by applying excitatory amino acids in the bath activating N-methyl-D-aspartate (NMDA) receptors and kainate receptors, but not quisqualate receptors. L-DOPA exerts a weak rythmogenic effect due to an action on kainate receptors. The kainate-induced rhythm is unchanged when a NMDA receptor antagonist is applied (2APV) and the N-methyl-aspartate-induced fictive locomotion can occur when kainate receptors are blocked (PDA). The burst frequency of the NMA-induced activity (dose range 30-5000 microM) is wide and ranges from 0.05-0.1 Hz up to 2.5-4 Hz, while the kainate-induced activity (dose range 7-30 microM) ranges from 0.5-1 Hz up to 4-8 Hz. This frequency range overlaps largely with that of the intact swimming animal. The findings further consolidate that NMDA receptors are efficient and demonstrates that kainate can also be effective in inducing fictive locomotion, and also that activation of either receptor type is sufficient. It has previously been shown that fictive locomotion elicited via sensory stimuli is depressed by NMDA and kainate receptor antagonists. It is suggested that these effects, presumably via aspartate and/or glutamate actions, are exerted on the input stage of interneuronal network.

Angiogenesis on the optic tectum of albino Xenopus laevis tadpoles.

Developing blood vessels were observed directly on the dorsal surface of the optic tectum of anesthetized, transparent albino Xenopus laevis tadpoles, stages 41-54. Case histories of individual tadpoles indicated that pial capillaries developed by the classical mechanism of sprouting of endothelial cells from existing blood vessels. 'Deep sources' appeared on the tectal surface during development. These were sites of upwelling blood cells from capillaries within the nervous tissue of the tectum into vessels on the surface. Few 'deep sinks' were observed in the dorsal tectum of normal tadpoles. The earliest deep sources were probably formed by sprouts from the surface vessels through the basement membrane and into the nervous tissue; later ones may also have formed from internal sprouts back to the surface. Maps of deep sources and of surface vessels in case histories indicated that neural tissue and blood vessels in the caudal half of the tectum grew faster than in the rostral half. The medial venules on the dorsal tectum originated as ordinary-sized rostrocaudal capillaries. They enlarged in diameter as they drained the increasing flow of blood from the tectum into the choroid plexus over the 4th ventricle. Some capillaries disappeared or regressed during development. Our observations on the tectum were consistent with the classical sequence of loss of flow, narrowing, collapse of the lumen, and retraction of endothelial cells into adjacent vessels. Likely sites for regression were upstream from a deep source and at crosslinks between transverse vessels on the lateral tectum. Morphometric parameters for tectal angiogenesis were (a) surface density (mm-1) calculated as total length of surface vessels divided by the dorsally projected surface area, and (b) density of deep sources (mm-2) calculated as total number divided by surface area. From stages 41/42 to 50 average surface density approximately doubled, and average density of deep sources increased about 5-fold. Some of the factors which might be expected to alter brain angiogenesis include nervous activity, availability of O2, and metabolic rate. Removal of one eye deprived the contralateral tectum of direct retinal inputs, while the ipsilateral side was a control in the same animal. Anterograde labeling of retinal axons with diI18 from the remaining eye confirmed projections only to the opposite side. No significant differences in densities of surface vessels or of deep sources were observed between the contralateral and ipsilateral sides of the tectum.(ABSTRACT TRUNCATED AT 400 WORDS)

LCBF changes in rat somatosensory cortex during whisker stimulation monitored by dynamic H2 clearance.

We evaluated increases in local cerebral blood flow (LCBF) localized to single activated cortical columns by H2 clearance methods. The rat whisker-barrel cortex is a model for cortical function and neural processing in active explorative behaviors. Up to four 30-40 microns Pt wire electrodes were inserted in or near the rat whisker-barrel cortex. Electrode positions were mapped by postmortem histology. H2 was generated electrochemically by constant current from one electrode and detected by one or more other electrodes 300-500 microns away. Changes in LCBF produced inverse changes in PH2. Shifts during steady H2 generation were calibrated against standard H2 inhalation clearance curves at rest and during inhalation of 7.5% CO2 for 1 min for quantitative estimates of LCBF. Contralateral whisker stimulation at 3 Hz, 1 min duration and delivered every 2 min produced the largest increases in LCBF. LCBF responses were detected in approximately 1 s. Stimulation of single whiskers produced the largest responses when an electrode was in the corresponding barrel. These results indicate that increased neural activity in a single cortical column produces blood flow responses primarily in that column.