Peter C. Johnson

Benzodiazepine binding in human brain: Characterization using [3H] flunitrazepam

[3H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [3H]flunitrazepam. The dissociation constant (KD) of [3H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [3H]-flunitrazepam binding invitro correlated well with their potency in several invivo human and animal tests. The density of [3H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.

Alterations in dopaminergic receptors in Huntington's disease.

To detect variations in dopaminergic receptors and cholinergic activity in regions of postmortem Huntingtons diseased brains, 3H-spiroperidol binding assays and choline acetyltransferase (ChAc) activities were carried out. A significant reduction in 3H-spiroperidol binding in the caudate nucleus, putamen and frontal cortex of choreic brains was detected which appeared to be due to a decrease in the total number of binding sites rather than to a decrease in affinity of 3H-spiroperidol for the dopaminergic receptor. In choreic brains, there were also significant reductions in ChAc activity in the caudate nucleus and putamen. The decreases of both 3H-spiroperidol binding and ChAc activity in the neostriatum suggest that the dopaminergic receptors are localized postsynaptically on cholinergic interneurons. Dopaminergic receptor alterations in the basal ganglia may be one of the causes of the abnormal motor movements found in HD while alterations of these receptors in the frontal cortex may be associated with the neuronal degeneration found in that area of choreic brains.

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Abstract Huntingtons Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntingtons Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [3H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [3H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntingtons Disease.

[3H]Pirenzepine identifies putative M1 muscarinic receptors in human stellate ganglia

The specific binding of [3H]pirenzepine ([3H]pZ) and [3H](--) quinuclidinyl benzilate ([3H](--)QNB) was investigated in homogenates of human stellate ganglia. [3H]pZ saturation isotherms yielded a K a of 14 nM and Bma x of 16.7 fmol/mg protein, while [3H](--)QNB binding curves yielded a K 0 of 59 pM and Bma x of 33.0 fmol/mg protein. This represents the greatest proportion of high affinity [3H]pZ labeling, relative to [3H](--)QNB, seen in any peripheral tissue examined thus far.The specific binding of [3H]pirenzepine ( [3H]PZ) and [3H](-) quinuclidinyl benzilate ( [3H](-)QNB) was investigated in homogenates of human stellate ganglia. [3H]PZ saturation isotherms yielded a Kd of 14 nM and Bmax of 16.7 fmol/mg protein, while [3H](-)QNB binding curves yielded a Kd of 59 pM and Bmax of 33.0 fmol/mg protein. This represents the greatest proportion of high affinity [3H]PZ labeling, relative to [3H](-)QNB, seen in any peripheral tissue examined thus far.

Dermal Nerves in Human Diabetic Subjects

The lack of a nerve tissue source that is easily, safely, and repeatedly available has been a major impediment to the study of human diabetic neuropathy. In this study dermal nerves from skin, obtained at biopsy and autopsy from the lower leg and at autopsy from the mid-abdomen, were subjected to quantitative electron microscopy to assess for diabetic perineurial cell basement membrane thickening, a change previously reported in sural nerve. A highly significant degree of perineurial cell basement membrane thickening was found in the diabetic subjects. Other structures in dermal nerves, such as axons, myelin, Schwann cells, and their organelles are also amenable to quantitative ultrastructural study. Skin biopsy is a way to obtain samples of peripheral nervous system tissue safely and repeatedly for the study of diabetic neuropathy.

The effects of some pharmacologically active amines on the rate of gastric emptying in rats.

Abstract The effects of several pharmacologically active amines on gastric emptying rate in male rats has been investigated. Of the compounds tested those with anticholinergic activity; amitriptyline, nortriptyline, imipramine, desmethylimipramine, 3-methylamino-1:1-diphenyl-prop-1-ene and its primary and tertiary amine analogues, dexamphetamine and methylamphetamine, all inhibited gastric emptying of a non-absorbable marker, polyethylene {1,2−14C} glycol. The compounds with little or no anticholinergic activity did not affect gastric emptying. The activity of the compounds in inhibiting gastric emptying falls in approximately the same order as their anticholinergic activity, lending support to the hypothesis that inhibition of gastric emptying is via in anti-cholinergic mechanism. The results also indicate that absorption is a prerequisite of activity in inhibiting gastric emptying and suggests that these compounds are not acting via a local mechanism.

Hematogenous metastases of carcinoma to dorsal root ganglia

SummaryHematogenous metastases of carcinoma to dorsal root ganglia was found in 2 of approximately 500 consecutive autopsies in which a lumbar dorsal root ganglion was routinely examined microscopically. The primary tumors were poorly differentiated colonic adenocarcinoma and oat cell carcinoma of the lung, both with widespread hematogenous metastases which spared the central nervous system. No symptoms were detected clinically. In the same series of patients the sural nerve as well as the lumbar plexus were histologically sampled but no examples of distant endoneurial metastases were found. The vascular endothelium of dorsal root ganglia is fenestrated and, presumably as a consequence, provides no bloodganglion barrier. This microvascular difference may account for the susceptibility of the ganglia to metastases when compared to nerve trunks which possess unfenestrated endothelium and blood-nerve barrier.

Effect of hyperbaric oxygen therapy or dimethyl sulfoxide on cerebral ischemia in unanesthetized gerbils.

To determine whether treatment with hyperbaric oxygen (HBO) or dimethyl sulfoxide (DMSO) could mitigate the fatal effects of cerebral ischemia, we anesthetized 68 gerbils with ketamine, ligated the right carotid artery (CA), and placed a snare occluder around the left CA. After 48 hours, 30 gerbils that were neurologically normal or had suffered only mild deficits were subjected to left CA occlusion without anesthesia for periods of 2 to 60 minutes. The onset of circling, posturing, falling, and lethargy began immediately; seizures and coma ensued 4 to 5 minutes later and persisted until release of the left CA occluder. All gerbils recovered after 2-minute staged bilateral CA occlusions. The mortality rate was 33% after both 5- and 10-minute occlusions and 100% after 20- and 60-minute bilateral occlusions. Twelve gerbils were placed in an HBO chamber (100% oxygen at 1.5 atmospheres) for 15 minutes during 20-minute bilateral occlusion; only 2 died (16% mortality rate). Thus, HBO therapy conferred significant protection against death from untreated ischemia (P less than 0.001). Histological examination showed that the extent of patchy bilateral ischemic neuronal damage was much less in surviving gerbils that received HBO therapy than in those that died after 20-minute occlusions. Fourteen gerbils were treated with DMSO, 2.5 g/kg intraperitoneally, during 5- or 10-minute bilateral CA occlusion; 12 died (86% mortality rate). Thus, DMSO provided no protection against fatal cerebral infarction; in fact, the results in the 10-minute reperfusion group suggest that DMSO may have a deleterious effect.

Relationships between microvascular function and capillary structure in diabetic and nondiabetic human skin

Despite the commonly held view that abnormalities in capillary morphology, in particular thickening of the capillary basement membrane, are partly responsible for diabetic ischemia, few studies have correlated anatomic and hemodynamic variables in the same diabetic subjects. In a previous study of 24 type II (non-insulin-dependent) diabetic subjects and 24 agematched control subjects, we showed that a standard finger exercise vasodilated cutaneous forearm vessels nearly equally (51%), but the postarteriolar flow responded differently between groups. Nondiabetic subjects increased flow by recruitment of capillaries, whereas diabetic subjects did so by capillary flow augmentation. Moreover, resting permeability-surface area product (PS) to pentetic acid was 85% higher in diabetic than nondiabetic subjects. In this study, these same subjects had their forearm skin biopsied and examined morphometrically by electron microscopy for capillary radius, basement membrane thickness, endothelial cell density, and a folding index of luminal membrane reduplication. All morphological variables were correlated stepwise in a saturated, analysis of covariance model with the physiological results. The correlations were sparse and specifically excluded basement membrane thickness. The highest r2 value was .432 between resting PS and a ratio of capillary density to endothelial cell number per capillary. These studies show little evidence that diabetic microvascular physiological variables are tightly connected to morphometric changes except for minor permeability changes, which rise with capillary density and decrease with endothelial cell number. Because PS to pentetic acid is increased in diabetic subjects at any level of capillary density, it seems reasonable that permeability may be increased above that of nondiabetic subjects. However, such a conclusion is tentative because anatomic measurement of capillary density gives only the maximum estimate of capillaries in the tissue, which may be greater than the number of capillaries with flow at the time of physiological measurement.

RNA content and volume of motor neurons in amyotrophic lateral sclerosis. I. The cervical swelling.

The content of RNA and the volume of motor neurons isolated from the lateral portion of the cervical swelling were examined in six control and six amyotrophic lateral sclerosis (ALS) cases obtained at autopsy. The mean volume of motor neurons in the ALS group did not differ significantly from the values obtained in the controls. However, in two cases of ALS with extensive neuronal loss and relatively long duration of disease most of the remaining cells were atrophic. The content of RNA in motor neurons averaged 300 micromilligram in the ALS group compared to 513 micromilligram in the control cases. The reduction in RNA content in the ALS group is approximately 42% and is statistically significant.