Per Lundborg

Postnatal development of bulk flow in the cerebrospinal fluid system of the albino rat: Clearance of carboxyl-[14C]inulin after intrathecal infusion

Car☐yl-[14C]inulin was infused into the spinal subarachnoid space of unanesthesized albino rats at 5, 10 and 30 postnatal days of age. Total cerebrospinal fluid volumes, estimated by infusions post mortem, were 122 ± 12 μl, 241 ± 24 μl and 250 ± 16 μl for 5-, 10- and 30-day-old rats, respectively. Measurements were made of steady-state rates at which inulin was removed from the cerebrospinal fluid system, constituting an estimate of the rate of cerebrospinal fluid formation. Values of 0.34 ± 0.03, 1.76 ± 0.18 and 1.88 ± 0.17 μl/min were found for 5-, 10- and 30-day-old rats, respectively. When inulin clearance was expressed as a percentage of the total fluid volume formed per minute, values of 0.28 ± 0.02, 0.73 ± 0.07,and0.75 ± 0.07 were found at 5, 10 and 30 postnatal days, respectively. Between 5 and 10 postnatal days of age, the total cerebrospinal fluid volume and rate of cerebrospinal fluid formation increased from 40 to 95% of 30-day-old values, correlating with morphological observations showing rapid maturation of the secretory epithelium of the choroid plexus. During early postnatal development, the formation of cerebrospinal fluid by the poorly differentiated columnar cells of the choroid plexus was greatly decreased and accompanied by sluggish bulk flow through the ventricles and subarachnoid spaces. It is hypothesized that, during this time, the inefficient ‘sink action’ of the cerebrospinal fluid system for extracellular space solutes in conjunction with an increased rate of entry of substances from the systemic circulation might increase the vulnerability of the developing brain to drugs and toxic agents.

Inhibition of catecholamine synthesis during ontogenic development.

Abstract The decrease with time of dopamine (DA) and noradrenaline (NA) in whole brain was analyzed in rats at 1, 4, 10, 14 and 21 days postnatal age following inhibition of tyrosine hydroxylase with H 44/68 (α-methyl- p -tyrosine-methylester) or of dopamine-β-oxidase with FLA-63 (bis[4-methyl-1-homopiperazinylthiocarbonyl]-disulfide). Over the time studied (up to 4 h after injection), the level of DA remained relatively constant at 1 day of age following treatment with H 44/68. At 4 and 10 days of age, there was a marked decrease in DA up to 2 h with no further change at 4 h, thereby producing a marked biphasic disappearance curve. At 14 and 21 days, the DA levels decreased continuously with time, but still with a slight biphasic tendency. NA decreased significantly from 1 day of age following treatment with H 44/68. After FLA-63, NA levels decreased at a much more rapid rate than after H 44/68 and DA accumulated to a slight degree. In a separate study on animals at 4 and 14 days of age, the decrease in DA and NA content was determined in brain regions 2 h after injection of H 44/68. At 4 days, a statistically significant decrease in DA content was noted only in the striatum, whereas at 14 days of age DA decreased significantly in all regions analyzed. NA decreased significantly at both 4 and 14 days in all regions except the striatum. The results indicate that NA-containing neurons become functional ( i.e. impulse conducting) at an earlier age than DA-containing neurons and also that DA-containing neurons present in the brain become functional at different postnatal ages.

Mechanisms for the elimination of 5-hydroxyindoleacetic acid from brain and cerebrospinal fluid of the rat during postnatal development

Abstract Mechanisms employed for the elimination of 5-hydroxyindoleacetic acid (5-HIAA) from brain and cerebrospinal fluid (CSF) were studied in rats during early postnatal development. Probenecid was used to inhibit the removal of 5-HIAA by active transport mechanisms. In the 1- and 4-day-old animals, the major route for efflux of 5-HIAA from brain to blood was indirectly via CSF pathways, and was mediated, presumably at the choroid plexus, by an active transport mechanism possessing an efflux rate sufficient to remove all the 5-HIAA formed in the immature brain. The slow rate of bulk flow of CSF could account for the removal of only a small percentage of the 5-HIAA formed in brain, and active transport of the catabolite from brain capillaries could not be demonstrated. In contrast, the elimination of 5-HIAA via CSF pathways was of minor importance in the 30-day-old rat. More than 75% of the major metabolite of 5-hydroxytryptamine was removed directly from brain to blood by an active transport mechanism, presumably located at the glia-capillary interphase.

Postnatal development of mechanisms for the elimination of organic acids from the brain and cerebrospinal fluid system of the rat: Rapid efflux of [3H]para-aminohippuric acid following intrathecal infusion

Abstract Mechanisms for the elimination of organic acids from the brain and CSF system were studied during early postnatal development, by infusing tracer doses of [3H]para-aminohippuric acid into the spinal subarachnoid space of unanesthetized albino rats at 5 and 30 days of age. The rate of bulk flow in the CSF system of the infant rat, as measured by the elimination of [14C]inulin, was extremely slow, reflecting the presence of relatively undifferentiated cells composing the secretory epithelium of the choroid plexus. Nevertheless, [3H]PAH was found to be rapidly eliminated by a mechanism of active transport which was inhibited by high concentrations of 5-HIAA and systemically injected probenecid. In contrast, a carrier-mediated transport system was not found in the CSF system of the 30-day-old rat, and the efflux rate for the organic acid could be accounted for entirely by a markedly facilitated rate of bulk flow. In the brain of 30-day-old rats, another seemingly independent mechanism for the rapid elimination of organic acids to the peripheral circulation was found. This efflux, presumably localized at brain capillaries, was mediated exclusively by active transport which was inhibited by 5-HIAA and probenecid. A mechanism for carrier-mediated transport was not found in the brain of the 5-day-old rat, although an equally rapid efflux of PAH occured as a result of diffusion through relatively undifferentiated cerebral capillaries.

Analysis of capillary and parenchymal aromatic l-amino acid decar☐ylase activity in regional brain areas during ontogenic development in the rat

Abstract The activity of aromatic l -amino acid decar☐ylase was measured in homogenates of brain tissue prepared from animals pretreated with the peripheral decar☐ylase inhibitor MK-486 (10 mg/kg) or saline 30 min before incubation. The brains were divided into 3 regions: hemispheres, diencephalon and brain stem. Developing animals were studied at 1, 4 and 21 days postnatal age, and adults at 180 days. The enzymic activity measured in saline-treated control tissue was considered representative of total decar☐ylase, and that measured in MK-treated tissue was considered representative of mainly neuronal decar☐ylase (parenchymal decar☐ylase) since brain capillary decar☐ylase is inhibited by MK-486. In all brain regions there was a gradual development of the parenchymal enzymic activity with age which changed the proportional composition of total decar☐ylase activity within the respective regions. The most rapid development of parenchymal decar☐ylase activity occurred in the brain stem, and the slowest in the hemispheres. The functional significance of a large proportion of capillary decar☐ylase in very young animals and the consequences of the development of parenchymal decar☐ylase with age were considered.

Digoxin-induced arrest of the cerebrospinal fluid circulation in the infant rat: implications for medical treatment of hydrocephalus during early postnatal life.

Summary: Three human infants with hypertensive obstructive hydrocephalus were treated with oral doses of digoxin beginning on the 2nd, 7th, and 13th week of postnatal life, respectively. One infant received 0.015 mg/kg/day and two infants were given 0.010 mg/kg/day of digoxin. The drug failed to alleviate any clinical signs of elevated intracranial pressure. Groups of 10-day-old infant rats were acutely digitalized by sc administration of three successive doses of digoxin (3 mg/kg body wt) at 20-min intervals. In the first group electrocardiograms and electroencephalograms were monitored. Additionally, arterial blood pressure was monitored by means of an aortic catheter. The electroencephalographic and cardiovascular response differed from controls only by minor changes in the ECG wave-form (increased amplitude of the QRS and T-waves). In the second group the rate of bulk flow [or cerebrospinal fluid (CSF) formation] was assessed by 5-min intrathecal infusion of 14C-inulin, performed 20 min after the last injection of digoxin.Comparative values for inulin clearance from the CSF system (µl/min) of experimental and control animals were calculated from inulin concentrations in CSF at stated intervals of 15-22, 22-30, 30-45, and 45-60 min after the last dose of digoxin. Rates of CSF formation were depressed to about 50% of control levels at 15 min, reaching a state of complete arrest between 22 and 45 min, and followed by rapid recovery to resting levels at 60 min. In the third experimental group of digitalized infant rats, concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in CSF and brain were assessed 80 min after the last injection of digoxin. Although no significant change in the amounts of 5-HIAA was found in brain tissue, the 5-HIAA concentrations in the CSF of digoxin-treated rats rose to values that were 140% greater than found in controls.An additional group of 10-day-old rats was killed 60 min after se injection of probenecid, 250 mg/kg body wt. Probenecid, a drug which competitively inhibits carrier-mediated transport of 5-HIAA from both the brain and CSF system, increased both brain and CSF concentrations of 5-HIAA by 55 and 432%, respectively.Speculation: We speculate that, although digoxin, in doses not toxic to the cardiovascular system, can completely arrest fluid secretion by the choroid plexus of the rat, its duration of action is too brief, rendering it useless for the treatment of chronic hydrocephalus in man.We also speculate that, despite the presence of efficient transport mechanisms localized to the brain parenchyma as well as the choroid plexus, the circulation of CSF is necessary for efficient elimination of acid metabolites from the central nervous system.

Transport mechanisms in the cerebrospinal fluid system for removal of acid metabolites from developing brain.

Postnatal development of a “blood-brain-barrier” for protecting the central nervous system from fluctuating concentrations of charged lipophobic molecules in blood, parallels the maturation of unique and highly efficient transport mechanisms for the intracranial removal of organic acids3,4. Prior studies have clearly delineated anatomic barriers called “tight junctions” in brain capillaries which prohibit penetration of proteins having molecular diameters larger than 20 A13. The permeability of this same barrier to smaller and more polar molecules has not been determined. Recent observations strongly suggest that homeostatic mechanisms designed to protect the internal milieu of brain may be viewed not only as a membranous barrier for blood-borne molecules based on their size, lipid solubility, and ionization constant, but as a series of selective filtration sites for carrier-mediated transport of watersoluble, polar molecules from the intracranial cavity into the systemic circulation13, 17.

PHARMACOLOGICAL APPROACHES TO MONOAMINE RECEPTORS DURING BRAIN DEVELOPMENT

Using biochemical, pharmacological, and behavioral methods, we have initiated studies on the ontogeny of functional catecholamine neurons in the brain. Studies in both rats and rabbits have suggested that receptors sensitive to dopamine develop over the first week or two of postnatal life. This period may represent a critical period for the development of these receptors in that chronic pharmacologic interference during the maturation of these receptors produces aberrant motor development. This evidence was interpreted to suggest that the drugs used interfered with dopamine receptor maturation and/or maturation of feedback control mechanisms in the synapse. Further, the evidence collected to date indicate that physiologically functional noradrenaline-containing neurons develop earlier than do dopamine-containing neurons. The differential rate of development of these neurons provides for a potential imbalance of released transmitter which may be of considerable significance. The studies we have presented have assisted in determining when the morphologic and enzymatic development of catecholamine pathways become integrated into functioning catecholamine neurons.

Postnatal development of mechanisms for the rapid efflux of primary amines from the cerebrospinal fluid system of the rat: Elimination of [3H]metaraminol following intrathecal infusion

Summary Tracer doses of [3H]metaraminol were infused into the spinal subarachnoid space of unanaesthetized albino rats at 5 and 30 postnatal days of age. The rates of elimination of this primary amine from the cerebrospinal fluid (CSF) system were compared with that of [14C]inulin in both age groups. In spite of the relatively undifferentiated state of the secretory epithelium of the choroid plexus and the sluggish rate of bulk flow, the elimination of metaraminol from the CSF system of the 5-day-old rat was extremely rapid. Moreover, this efflux from CSF to blood was mediated almost exclusively by a mechanism of active transport which was both saturable at high concentrations and competitively inhibited by the quaternary amine, N1-methyl-nicotinamide. Although an extremely rapid rate of elimination of metaraminol was also found in the older animal, a carrier mechanism for active transport could not be demonstrated, and the efflux mechanism could be accounted for entirely by a markedly facilitated rate of bulk flow. Hence, during development of the CSF system, the mechanism of bulk flow provides an additional and perhaps less vulnerable route for the elimination of amines from the CSF.