Characterization of the binding of a morphine (mu) receptor-specific ligand: Tyr-Pro-NMePhe-D-Pro-NH2, [3H]-PL17.

The highly mu-receptor-selective ligand Tyr-Pro-NMePhe-D-Pro-NH2 (PL17) was custom tritiated and its binding to rat brain membranes was directly measured. The data were well fit by a model assuming the existence of a single homogeneous population of binding sites. Scatchard analysis yielded values for Kd and binding sites of 6 nM and 0.16 pmol/mg, respectively. As expected for binding to an opiate receptor, addition of sodium, magnesium, and guanyl nucleotides to the assays resulted in a modulation of ligand binding. In all cases tested, however, no significant deviations of the Scatchard plots from linearity were observed. Furthermore, displacement of [3H]-PL17 by all opioid ligands tested was monophasic, consistent with simple competitive inhibition at a single binding site. The IC50 values thus obtained showed good agreement with previously published values determined using less selective radiolabeled ligands. The regional distribution of [3H]-PL17 binding to rat brain was examined by in vitro autoradiography. The labeling pattern was similar to that seen for other mu-type ligands. Because of its high selectivity, the binding of [3H]-PL17 to the mu-type receptor can be measured directly without the need to suppress binding to other sites. As such, [3H]-PL17 should be a useful ligand for dissecting the actions of multiple opiate receptors.

Single unit activity in frontal cortex and caudate nucleus of young and old rats.

Spontaneous neuronal activity was recorded extracellularly from isolated single units in frontal neocortex and caudate nucleus of young and aged F344 rats anesthetized with urethane. Average firing rates, mean interspike intervals (ISI) +/- standard deviations, and ISI frequency histograms were computed and analyzed by microprocessor. For frontal cortex cells (N = 226), there was a nonsignificant trend toward slower average discharge rates in the old group. However, a significantly longer mean ISI and proportionally more very slow firing cells (less than 1 Hz) were observed in old rats. A laminar analysis of frontal cortex unit activity in young animals showed average discharge rates to be distributed somewhat evenly throughout the cortical mantle with the exception of the zone 1200-1400 mu beneath brain surface. This depth corresponds approximately to layer V where a 50% increase in mean firing rate in young animals was observed. In aged animals, this increased cell firing in layer V was absent, while mean discharge rates in other laminae remained essentially the same in the young and old rat groups. Caudate nucleus cells (n = 70) showed a significant shift towards fewer fast discharging cells in old rats, with the average firing rate diminished by one-third. Although more brain regions need to be examined in a similar fashion, the consistency of the present results with those previously reported for the brainstem and cerebellum suggests that slower firing rates and longer ISIs are likely to be wide-spread throughout the brains of aged rats.

Developmental protein malnutrition in the rat: effects on single-unit activity in the frontal cortex.

This study evaluated the effects of developmental protein malnutrition on the spontaneous electrical activity of frontal cortex neurons in the anesthetized rat. Rats were raised prenatally and postnatally on either an 8% or 6% casein diet until adulthood. Compared to the 25% casein controls, both malnourished groups showed a 30-36% decrease in mean discharge rates and a 100-200% increase in the percentage of cells with very slow (less than 1/s) discharge rates. Most of the diet-related changes were confined to a zone 600-1200 micron below the brain surface, approximately cortical layers III, IV and V. A second set of studies in which diet reversals were introduced at birth or in adulthood found that: (a) restoration of a normal 25% casein diet at birth did not appreciably attenuate the effect of prenatal administration of an 8% casein diet; (b) introduction in adulthood of the 8% casein diet to a normally fed rat had no effect; (c) introduction of the 8% diet at birth, however, produced effects in adulthood comparable to those seen when the protein malnutrition was introduced in the prenatal period. Thus, the rat brain is sensitive to both prenatal and postnatal protein malnutrition (starting at birth). Most importantly, the effects of prenatal protein malnutrition on the activity of frontal cortex neurons do not appear to be reversible by restoration of a normal diet in adulthood or at birth.

Horseradish peroxidase labeling of extracellular single unit recording sites.

Horseradish peroxidase (HRP) has been widely used in neurobiology to trace neural pathways (axonal transport) and to correlate physiology with morphology (intracellular injection). We report that 5% HRP in 0.1 M phosphate buffered normal saline may be used to fill micropipettes for stable extracellular single cell recordings. HRP can then be iontophoresed at the desired recording site(s) and does not appear to impair activity of other neurons in the area or to cause damage to the electrode tip after the marking procedure. Subsequent perfusion, sectioning and reacting of the brain with the diamino-benzidine chromagen yielded a discrete (100-500 mu diameter) brown reaction product in the extracellular space, as well as peroxidase filled perikarya which were readily distinguishable from damaged vascular or neural elements. This method is highly reliable and provides a simple technique for localizing the tip of micropipette electrodes.

Spontaneous forebrain neuronal activity in developmentally protein malnourished rats.

The characteristics of the spontaneous discharges of populations of single neurons in the neocortex and thalamus of rats reared under chronic protein malnutrition (8% casein) or a normal diet (25% casein) were evaluated. Results from 700 neurons showed the malnourished rats had fewer fast firing cells, an overall lower discharge rate and an altered firing pattern (fewer cells which exhibited bursting activity). These findings are the first demonstration of altered neuronal discharges in the forebrains of adult malnourished rats.

Differential uptake of peroxidase (HRP) and peroxidase-lectin (HRP-WGA) conjugate injected in the nodose ganglion of the cat.

A comparison was made of the uptake and consequent axonal transport of peroxidase and peroxidase-lectin conjugate injected in low concentrations (0.167%) in the nodose ganglion of cats. At the light microscopic level horseradish peroxidase (HRP)-wheat germ agglutinin (WGA) intensely labeled only central terminal fields of vagal afferents (anterograde), while free HRP only labeled perikarya in vagal motor nuclei (passing retrograde). Low concentrations of these proteins, in addition to normal diffusion equilibria, permit a differential distribution of those species demonstrating some affinity for cell membranes. We attribute these differences in the uptake of HRP and HRP-WGA to the selective affinity of WGA for cell surface receptors (n-acetyl glucosamine) on the plasma membrane. This results in a greater number of WGA molecules coupled to HRP being internalized in any given endocytotic event compared to free HRP. The fractionation of efferent and passing fiber populations within a nodosal injection site can be discriminated with these different preparations.