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The topic of offender DNA databases and the retention of offender's DNA samples are issues that continue to generate debate between privacy advocates and forensic DNA scientists. Considered previously by [...]

[3H]CP-55,940, a high-affinity cannabinoid receptor ligand, was used for in vitro binding and autoradiography in peripheral tissues in the rat. Specific cannabinoid receptor binding was found to be restricted to components of the immune system, i.e., spleen, lymph nodes and Peyer's patches. Displacement studies showed that this binding is identical (similar Kd and structure-activity profile) to that in brain. Cannabinoid receptors in the immune system are confined to B lymphocyte-enriched areas, i.e., the marginal zone of the spleen, cortex of the lymph nodes and nodular corona of Peyer's patches. Specific binding is absent in T lymphocyte-enriched areas, such as the thymus and periarteriolar lymphatic sheaths of the spleen. Certain macrophage-enriched areas, i.e., liver and lung, lack specific binding. Thus, the single peripheral cell type that may contain cannabinoid receptors is the B lymphocyte. Numerous sites have dense binding that could not be displaced by excess unlabeled drug. These nonspecific sites were found in the liver, adrenal glands and sebaceous glands, which are high in fat content, and in the heart, pancreas, components of the male and female reproductive systems and the epithelium of the esophagus. Thus, the highly lipophilic nature of cannabinoids does not appear to be the sole determinant of nonspecific binding. The data suggest that cannabinoids may exert specific receptor-mediated actions on the immune system of rats. Perhaps, also at high concentrations, cannabinoids exert membrane effects at sites where they are sequestered nonspecifically.

Slide-mounted sections of unfixed frozen rat brain can be labeled in vitro with [3H]naloxone to show the mu-like ligand selectivity characterized in previous studies. We have developed an autoradiographic technique using hot paraformaldehyde vapors to prevent diffusion of ligands with reversible binding. Resolution at the light level is sufficient to detect concordance between receptor patterns and terminal fields of axonal projections marked by tract-tracing techniques. The opiate receptor distribution suggests the existence of widespread intrinsic and several longer multisynaptic "opiatergic pathways within sensory and limbic circuits. One multisynaptic pathway may link olfactory structures with limbic circuits in the amygdala and habenula. Another may lie in limbic cortical structures. Opiate receptors are numerous also in sensory systems, and within primary sensory nuclei (visual, auditory, olfactory, somatic) they are found superficially in laminated structures. Together, the opiate receptors are well placed to control incoming sensory and subsequent limbic information processing.

The mechanism by which mu ligands inhibit the binding of prototypic delta agonists to preparations of brain membranes is controversial. Most investigators assume competitive inhibition. In this study, we examine the interaction of the mu agonist oxymorphone and delta agonist DSTLE (D-Ser2-Thr6-Leu-enkephalin) with [3H]D-Ala2-D-Leu5-enkephalin (DADL) binding to membranes of rat brain. According to the two-site competitive model, mu ligands are competitive inhibitors at both sites. The two-site allosteric model supposes that mu ligands are competitive inhibitors at one binding site, and noncompetitive inhibitors at the other binding site. Quantitative analysis of DSTLE and oxymorphone binding demonstrated that the two-site allosteric model fit the data significantly better than did the two-site competitive model, and that oxymorphone is a noncompetitive inhibitor of the lower affinity [3H]DADL-binding site. Autoradiographic studies demonstrated that the lower affinity [3H]DADL-binding site (mu-noncompetitive binding site) had an anatomical distribution apparently indistinguishable from that obtained with [3H]oxymorphone (type I pattern), supporting the hypothesis that the lower affinity delta-binding site is the delta-binding site of an opiate receptor complex consisting of interacting mu- and delta-binding sites.

The binding of a radiolabeled "mu receptor" prototype opiate, dihydromorphine (H2morphine), and the binding of a "delta receptor" prototype, [D-Ala2,D-Leu5]enkephalin (D-Enk), to slide-mounted rat caudate slices were simultaneously compared quantitatively and visualized by autoradiography. Generally, D-Enk bound to opiate receptors distributed evenly throughout the entire striatum (type 2 pattern), whereas H2morphine labeled discrete islands or patches of receptors (type 1 pattern). In the presence of Mn2+ (3 mM) or other divalent cations, however, Na+ and GTP at 25 degrees C caused an increase in D-Enk binding at the expense of H2morphine binding at striatal opiate receptor patches. Thus, these conditions shifted D-Enk binding from an even pattern to one that included both an even and patchy distribution. These incubation conditions not only promoted D-Enk binding to striatal patches but also enabled the opiate receptor to regulate adenylate cyclase with the same (P less than 0.01) ligand selectivity pattern as that obtained by the displacement of D-Enk binding. The relative affinity of opiate receptors in striatal patches for opiate peptides, naloxone, and morphine appears to be a function of incubation conditions and coupling to adenylate cyclase and is not indicative of distinctly different opiate receptors. We postulate a three-state allosteric model consisting of mu agonist-, mu antagonists-, and adenylate cyclase-coupled delta-agonist-preferring states, whose equilibrium may be regulated by a sulfhydryl group mechanism.

Expression of neuropeptide messenger RNAs in striatal neurons was studied in post mortemhuman brain tissue by the use of in situhybridization histochemistry. Clusters of cells expressing high levels of prodynorphin messenger RNA, and less strikingly, preprotachykinin messenger RNA, were prominent in the caudate nucleus and were present but less pronounced in the putamen. Proenkephalin and prosomatostatin messenger RNA-containing cells were more homogeneously distributed throughout the striatum, though the latter were much sparser. The four neuropeptide messenger RNA patterns in the nucleus accumbens were rather homogeneous compared with the dorsal striatum. Of these, prodynorphin messenger RNA showed a higher level of expression per cell in the nucleus accumbens relative to the dorsal striatum. The relationship of neuropeptide-containing cell clusters to the striosomal organization was characterized by looking at the register of these markers with patterns of low acetylcholinesterase activity and dense μ opiate receptor binding. In the caudate and putamen, clusters of cells expressing high levels of dynorphin and preprotachykinin messenger RNAs were clearly in register with the striosomes. The accumbens was defined by high prodynorphin messenger RNA levels, both low and high levels of acetylcholinesterase staining, and very low to absent μ opiate receptor binding.

LKB film autoradiography of 2-]3H]deoxy-D-glucose uptake shows that ketamine, administered in anesthetic doses, alters the pattern of metabolic activity in rat hippocampus. The labeled metabolic marker can be washed out of the slide-mounted tissue sections by preincubation to permit in vitro autoradiography of drug and neurotransmitter receptors in the same animal. In this way, opiate and phencyclidine receptor distributions may be correlated with patterns of glucose utilization in adjacent sections. If the observed relative enhancement of 2-deoxy-D-glucose uptake in the stratum moleculare of hippocampus reflects elevated metabolism in nerve terminals there, then the binding of ketamine to phencyclidine receptors on neurons in distant afferent sites, such as entorhinal cortex, may initiate the physiologic and metabolic effects.

[3H]Phencyclidine ([3H]PCP) binds specifically to an apparently single class of binding sites on slide-mounted sections of rat olfactory bulb (Kd = 46 nM; Bmax = 10.5 fmol per slice). Bound [3H]PCP can be displaced by nonradioactive PCP and a series of its analogs with relative potencies that correlate closely (P less than 0.001) with values determined in a rat discrimination test that utilized PCP as a cue. Although morphine, naloxone, and opiate peptides do not displace bound [3H]PCP, psychotomimetic benzomorphans, classed as "sigma opiates," are quite potent displacers in vitro and have PCP-like behavioral properties in vivo. These results suggest that phencyclidine and the sigma opiates act at the same sites. [3H]PCP binding sites were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry and color coding. The [3H]PCP binds most densely to cortical areas, diffusely in neocortex, and somewhat heterogeneously in the laminae of the hippocampal formation and dentate gyrus. Most of the brainstem and spinal cord show low specific [3H]PCP binding, with gray matter generally showing more binding than white.

The inflammatory cytokine interleukin-1 has been implicated as a mediator of many centrally controlled responses, such as fever and increased activity of the hypothalamic-pituitary-adrenal axis, after systemic infections. To identify the neuroanatomical loci of brain interleukin-l-producing cells during infection, we investigated interleukin-1β messenger RNA expression by in situ hybridization histochemistry using a 500 nt ribonucleotide probe applied on brain sections from rats injected intraperitoneally with 2.5 mg/kg bacterial lipopolysaccharide or saline. In control animals, interleukin-1β messenger RNA was not detectable. In the brains of lipopolysaccharide-injected animals, two temporally and spatially distinct waves of interleukin-1β messenger RNA induction were observed. First, cell labelling appeared at 0.5 h, peaked at 2 h, and declined at 4–8 h. The labelled cells were concentrated in circumventricular organs - organum vasculosum of the lamina terminalis, subfornical organ, median eminence and area postrema - and in choroid plexus, meninges, and blood vessels. Second, at 8–12 h, scattered small cells became labelled throughout the entire brain parenchyma; the labelling subsided by 24 h. Labelling was not observed in any neurons. In the pituitary, lipopolysaccharide induced strong interleukin-1(3 messenger RNA expression initially in the anterior lobe at 0.5-1 h, and later in the neural lobe at 1–2 h, and subsiding thereafter. The results show that at early time points, peripheral lipopolysaccharide induces interleukin-1β message production at the blood brain barrier and in circumventricular organs where the blood-brain barrier is leaky. After a time delay of 6–10 h, however, interleukin-1β messenger RNA is primarily expressed by non-neuronal cells of the brain in the brain parenchyma.