Carl M. Rovainen

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.

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

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

Stimulation- and calcium-dependence of vesicle attachment sites in the presynaptic membrane: a freeze-cleave study on the lamprey spinal cord.

Abstract Vesicle attachment sites (VAS) were identified in presynaptic profiles of aldehyde-fixed, freeze-cleaved spinal cord of the sea lamprey, Petromyzon marinus . The numbers of VAS per profile increased significantly after incubation in high K in the presence of Ca, but not in high K, Ca-free fluid. Similarly, electrical stimulation increased the number of VAS above control values. Plasmalemmal vesicles were observed much less frequently, but they also increased after the two types of stimulation. These results support the hypothesis that VAS represent a morphologically distinct stage in the release of transmitter. Other structures identified were unique large particles in glial membranes bordering the surface of the cord and gap junctions at some synapses of large axons.

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

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

Ministrokes in Rat Barrel Cortex

BACKGROUND AND PURPOSE Many stroke models in rats are based on occlusion of the middle cerebral artery, which supplies a significant portion of multifunctional cortical and deep structures in the cerebral hemisphere. The purpose of this study was to develop a model for direct observation in real time of blood flow in and around focal ischemic regions of the cortex of known function. METHODS Cranial windows were placed over the parietal cortex of adult Wistar and Sprague-Dawley rats anesthetized with ketamine and xylazine. Whisker barrel cortex responding to stimulation of the contralateral whiskers was identified by an intrinsic optical signal. Transits of vital dyes were recorded by videomicroscopy before and after ligation of three to six branches and major collaterals of the middle cerebral artery through the dura. Infarcts were demonstrated with triphenyl-tetrazolium chloride staining; their relation to barrel cortex was determined by Nissl and cytochrome oxidase histology. RESULTS Reduced blood flow in small ischemic regions was outlined by patient blue violet in the surrounding nonischemic area; arteriovenous latencies increased more than four times in ischemic cortex. Infarcts,typically 3 mm or less, were seen at 24 hours in 8 of 16 Wistar and 9 of 9 Sprague-Dawley rats. The ministrokes were confirmed by histology to be in the somatosensory cortex. CONCLUSIONS This model of local ischemia, produced deliberately in the functionally defined barrel cortex in rats, leads to ministrokes. Changes can be followed by videomicroscopy as they develop, and processes of recovery can potentially be monitored. Infarcts are confirmed by histology for their location and extent in the somatic representation.

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.

Feeding and Breathing in Lampreys

A basic problem faced by the agnathans in evolution was how to feed and breathe without jaws. Three solutions are represented by lampreys and their ammocoete larvae, reviewed here, and hagfishes. Lampreys feed upon fish with their suckers and breathe in and out of their branchial gill sacs. Parasitic species of lampreys can be flesh-feeders or blood-feeders, depending primarily on the structure of their teeth. Feeding behavior is characterized by rhythmic rasping, negative pressure pulses in the sucker, and swallowing of fluid into the gut. Ammocoete larvae use a velar pump for unidirectional ventilation and suspension feeding. In both lampreys and ammocoetes the branchial basket is actively compressed for exhalation; branchial expansion and inhalation is by passive elastic recoil, but in ammocoetes water is drawn from the mouth. Central pattern generators for respiration are distributed in the medulla, particularly lateral to Vm, and drive branchial motoneurons in VIIm-IXm-Xm. Trigeminal pattern generators in lampreys may be a holdover from the ammocoete stage, in which they drive nearby velar motoneurons as the primary pump for ventilation. Respiration in lampreys and ammocoetes is stimulated by hypoxia and modulated by reflexes. Metamorphosis from ammocoete to adult lamprey involves extensive remodeling of the head with regression and replacement of most muscles. Trigeminal motoneurons are probably preserved during metamorphosis, as inferred from constant maps of motoneurons in Vm. This hypothesis is supported by analogy with anuran metamorphosis in which V motoneurons are retained and remodeled. In Mallatts current models, the earliest vertebrates breathed by branchial contractions and valves; jaws initially evolved for better ventilation and later were used for feeding.

Structure and chemistry of glucose-producing cells in meningeal tissue of the lamprey.

Abstract The structure of meningeal tissue of sea and brook lampreys was investigated with light and electron microscopy. Glucose and other metabolites were measured in separated meningeal and nervous tissues, and a direct fluorometric method was developed to follow the release of glucose from incubated meningeal tissue. Meningeal tissues around brains and spinal cords of both larval and adult lampreys of both species contain prominent round cells. These cells are the probable source of the glucose produced since they contain considerable glycogen. They also contain lipid droplets, darkly staining mitochondria, membrane-bound granules, and whorled endoplasmic membranes which are related to glycogen deposits. Direct extracellular pathways extend from round cells to the basement membrane covering the brain. Evidence is presented that glucose produced by the meninges can pass into the brain. Furthermore, dyes injected into the meningeal tissue also enter the nearby nervous tissue. Kinetics and stability of meningeal glucose-6-phosphatase, which probably participates in the conversion of glycogen to glucose, were investigated. The enzyme resembles that of mammalian liver and kidney, but differs from β-glycerophosphatase.

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.

Respiratory motoneurons in lampreys

Summary1.Motoneurons to muscles of the branchial basket were identified by physiological critera in isolated brain-gill preparations of small adult lampreys,Petromyzon marinus andIchthyomyzon castaneus. Intracellular stimulation of motoneurons produced one-to-one contractions in ipsilateral motor units; one-to-one electromyographic potentials were also observed (Fig. 2). Motoneurons were confirmed as such by antidromic stimulation (Fig. 3).2.Identified respiratory motoneurons were located in the IX and X cranial motor nuclei, through which they were distributed somatotopically (Fig. 1). Cells innervating the branchial constrictor, diagonal, gill pouch, and valve muscles were intermixed. Motoneurons marked by intracellular dye injection (Fig. 4) had the same morphology as other cells of the rostral motor nucleus of the vagus.3.During respiratory movements branchial motoneurons were driven by a periodic and powerful burst of excitatory postsynaptic potentials (EPSPs) (Fig. 5). Several observations indicated that these EPSPs were not due to electrical coupling of motoneurons, but rather were produced by unidentified pacemakers in the brain.