Collagenase immunolocalization in cultures of rheumatoid synovial cells.

Cultures of rheumatoid synovial cells that have been enzymatically dissociated and are adherent to a culture vessel are morphologically heterogeneous. When these cells are cultured on a collagenous substrate for 2 to 6 days at 37 degrees C in serum-free medium, they produce collagenase. A monospecific antibody to human collagenase has localized the enzyme extracellularly around cytoplasmic extensions of dendritic cells and intracellularly within a few macrophage-like and fibroblast-like cells.

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.

3H-estradiol distribution in female, androgenized female, and male rats at 100 and 200 days of age.

Tissue distribution of radioactivity was studied 48 h after gonadectomy and 1 h after iv injection of 3H-estradiol (1 mug/kg body wt) in 100- and 200-day-old normal female rats, female rats androgenized with 30, 100 or 1250 mug testosterone propionate (TP) at 5 days of age, and male rats. Receptor-mediated uptake of estradiol, as shown by diethylstilbestrol (DES) competition, was highest in preoptic area-anterior hypothalamus (POA-AH) and median eminence-basal hypothalamus (ME-BH), but also occurred in dorsal hypothalamus, pre-hypothalamic area, amygdala and septum in all groups. At 100 days of age there were no differences in brain radioactivity levels between females and androgenized females or males. At 200 days of age radioactivity levels in POA-AH and ME-BH tended to be lower in androgenized female and male rats than in normal females. Also, radioactivity levels in the amygdala were lower in the 1250 mug TP-treated females than in normal females. When expressed per unit fresh weight, uptake in the anterior pituitary tended to be lower in androgenized rats at 100 days of age and was higher in males at 200 days of age than in normal females, but did not differ among any of the groups when expressed as uptake per organ. Thus, the well-known differences among these groups in neural regulation of gonadotropin secretion and sex behavior were not correlated with consistent differences in specific estradiol binding by hypothalamic or other brain areas or pituitary. The uterus took up less estradiol in androgenized females than in control females at both ages. Estradiol receptor activity was demonstrated in kidney of all animals and in seminal vesicles. At both ages radioactivity levels in the toluene and ethanol extracts of lever and kidney were strikingly higher in males than in the female groups. Reviewing the data, it appears that despite some evidence of differences between groups, males, females and androgenized females all have relatively similar limited-capacity, estradiol-uptake systems in brain and pituitary, as measured under the conditions of the present study.

Catecholamine synthesis inhibitors acutely modulate [3H]estradiol binding by specific brain areas and pituitary in ovariectomized rats.

Drugs known to alter endogenous levels of catecholamines were administered to adult ovariectomized rats to assess catecholaminergic effects on estradiol (E2) uptake and binding in nuclear and supernatant fractions of pituitary and specific brain regions and on cytoplasmic E2 receptor numbers and affinities. Specific (i.e. diethylstilbestrol-blockable) binding in vivo was measured 1 h after the iv injection of [3H]E2 (1 micrograms/kg). Administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (alpha MPT) 2 h before [3H]E2, to reduce levels of dopamine (DA), norepinephrine (NE), and epinephrine (Ep), decreased total and specific [3H]E2 binding by 36-56% in the nuclear fraction of the anterior pituitary, basal hypothalamus, and anterior hypothalamus. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDC), administered 2 h before [3H]E2 to reduce levels of only NE and Ep, increased the total and specific uptake of [3H]E2 by 62-140% in nuclear and supernatant fractions of the anterior pituitary and also increased uptake in several brain areas. In vitro analysis of hypothalamic and pituitary cytoplasms showed that in vivo administration of DDC increased E2 binding. Scatchard analysis showed that DDC increased receptor numbers 18-29%, with no change in the dissociation constant in pituitary cytoplasms. At the same time, plasma PRL levels were reduced by DDC treatment, indicating that DDC had increased DA output. Phenoxybenzamine (a blocking agent at alpha 1 postsynaptic binding sites) and a high dose of clonidine (a pre- and postsynaptic alpha-receptor agonist) did not significantly alter specific uptake in the cell nuclear fraction of any tissue, suggesting that postsynaptic alpha-receptors do not play a major role in modulating [3H]E2 uptake. No drug altered plasma levels of radioactivity. Because alpha-methyl-p-tyrosine and DDC both inhibit synthesis of NE and Ep, it is suggested that their opposite effects on uptake of [3H]E2 are related to their opposite effects on DA output. This interpretation is compatible with our previous observations that DA agonists increase [3H]E2 uptake in brain and pituitary in ovariectomized rats.

Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.

Matrix metalloproteinases (MMPs) together degrade virtually all the components of the extracellular matrix and are likely to play a role in remodeling of endometrial tissue during the normal menstrual cycle. Primary cultures of human endometrial stromal cells secreted a number of MMPs. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were measured in culture medium by specific enzyme assays. Production of the enzymes did not correlate with the time of the menstrual cycle at which the tissue was collected. Identities of MMP-1 and MMP-3 were confirmed by Western blots, by comparison of mol wt with those of purified enzymes on casein zymography, and by inhibition of these activities with EDTA and 1,10-phenanthroline. Northern analysis demonstrated specific messenger ribonucleic acid for pro-MMP-1 and pro-MMP-3 in phorbol myristate acetate-stimulated stromal cells. Two gelatinases were detected by gelatin zymography: MMP-2 (gelatinase-A) was present in two forms (72 and 67 kilodaltons), and MMP-9 (gelatinase-B) was present as a homodimer with a mol wt of approximately 180 kilodaltons. MMP-9, but not MMP-2, secretion was stimulated by phorbol myristate acetate. All enzymes could be activated in vitro by (4-aminophenyl)mercuric acetate. Both interleukin-1 alpha and tumor necrosis factor-alpha stimulated the secretion of MMP-1, MMP-3, and MMP-9, but not MMP-2, from the cells in a concentration-dependent manner. MMP production by endometrial stromal cells has a potentially important role in the processes of menstruation and implantation.

Morphometric effects of exposure to lead during the preweaning period on the hippocampal formation of aging rats.

Our previous morphologic studies in the rat demonstrated that exposure to lead during the preweaning period induced delays in the maturation of late-developing regions of the hippocampal formation at 15 days of age, followed by normal development or hypertrophy of the same areas in young adulthood. The present study was carried out to determine whether or not subtle or latent effects of such exposure to lead may be unmasked with the additional challenge of aging. To do this, mid-dorsal sections of the hippocampal formation from middle-aged (578-631 days old) Long-Evans control rats and from rats exposed to lead from birth until weaning via dams drinking 0.2% lead acetate were analyzed by light and electron microscopy. Exposure to lead did not alter areas of either neuropil or neuronal layers of the hippocampus or the dentate gyrus or the numbers per section or numerical densities (numbers per unit area) of neurons in hippocampal CA3 stratum pyramidale or dentate stratum granulosum. It did reduce mean size of complex invaginated mossy fiber synapses without altering their numbers in the proximal (close to dentate gyrus) mossy fiber zone, which was the zone also affected at 15 and 90 days of age in our previous studies.

Catabolism of intact fibrillin microfibrils by neutrophil elastase, chymotrypsin and trypsin.

We present ultrastructural and biochemical evidence for the turnover of intact fibrillin microfibrils by the serine proteinases, neutrophil elastase, chymotrypsin and trypsin. Rotary shadowing electron microscopy revealed that serine proteinase treatment of intact microfibrils isolated from foetal bovine skin resulted in extensive degradation. Microfibrils were destroyed by neutrophil elastase and effectively disrupted by chymotrypsin and trypsin, with no morphologically identifiable arrays remaining. Evidence of defined fibrillin degradation products was obtained by Western blotting of these enzyme-treated fibrillin assemblies. Fibrillin immunoprecipitated from dermal fibroblast culture medium was also comprehensively degraded by these enzymes. These observations demonstrate that serine proteinases are potent effectors for the physiological and pathological catabolism of microfibrils, and suggest a key role in elastic fibre degradation.

Host-mediated effectors of tumor invasion: role of mast cells in matrix degradation.

The role of collagenolytic enzymes in tumor invasion and metastasis has been emphasized, but the source of enzyme activity has remained unclear. Degradation of stromal connective tissue is a common feature of invasive neoplasia, and host-tumor cell interactions are probably important for localized collagenolysis. We have examined the role of mast cells in malignant cell invasion using cells derived from the rat mammary adenocarcinoma 13762NF. Histologic studies have shown increased numbers of mast cells at the zone of tumor invasion. Mast cell products and conditioned medium from such cells stimulated the production of collagenolytic enzymes by stromal fibroblasts as well as certain subpopulations of tumor cells in vitro. The tumor cell response to mast cell-mediated stimulation of collagenolysis appears to be related to the metastatic potential of the tumor cell. A subpopulation of host fibroblasts derived from the invading tumor zone was also found to be more responsive to mast cell factors than normal fibroblasts, as judged by collagenase production. Thus the mast cell has the potential to induce collagenolytic activity from both host fibroblasts and tumor cells.

Neovascularization in early atherosclerotic lesions of human carotid arteries: its potential contribution to plaque development.

Neovascularization is a prominent feature of late-stage atherosclerotic lesions and their complications but is generally regarded as an insignificant, undetectable component of the earliest stages of plaque development, probably because of relatively poor histological techniques. Using an improved vascular staining procedure, we have examined the extent of neovascularization in the earliest plaque lesions. Combined monoclonal antibodies to CD31, CD34, and von Willebrand factor have provided an ultrasensitive technique with which to visualize blood vessels in early atherosclerotic lesions (n = 55) of human carotid arteries obtained through surgical procedures. Capillary-like microvessels were shown in very early atherosclerotic lesions (type II), where they were associated with the distribution of macrophages and a few immature mast cells. Neovascularization was more prominent in type III lesions with vessels of variable size, often providing a focus around which local accumulations of macrophages and apolipoproteins A-I and B were visualized. Thickened type III lesions usually showed an intricate network of microvessels, together with numerous mast cells. These studies have shown neovascularization as a prominent feature of early stages of atherosclerotic plaque development. Whereas distribution of apolipoproteins A-I and B were observed in the very earliest stages of the plaque intima, these lipids, together with macrophages, foam cells, and mast cells, were observed as perivascular accumulations in a proportion of type II and III lesions. Such findings indicate that neovascularization is an important feature of early plaque development and may provide an additional or alternative source of leukocyte and lipid accumulations relative to the arterial lumen.

Menstruation: induction by matrix metalloproteinases and inflammatory cells.

Menstruation occurs at the end of a normal reproductive cycle in the human female, following the fall in progesterone resulting from the demise of the corpus luteum. Current data support a central role for the matrix metalloproteinases in menstruation but their focal pattern of expression within peri-menstrual and menstrual endometrium suggests local rather than hormonal regulation. This review emphasizes the similarities between menstruation and an inflammatory process and examines the relationship between cells of hemopoietic lineage, particularly mast cells, eosinophils, neutrophils and macrophages, and the local production and activation of matrix metalloproteinases within the endometrium. It proposes a complex of critical regulatory circuits, initially activated by the withdrawal of progesterone, which provide interactions between the migratory cells that produce a myriad of important regulatory molecules and endometrial stromal and epithelial cells which produce both chemokines and matrix metalloproteinases. These mechanisms could account for the focal nature of the tissue degradation at menstruation.

Immunolocalisation studies of matrix metalloproteinases-1, -2 and -3 in human melanoma.

The matrix metalloproteinases (MMPs) are considered to have an important role in connective tissue degradation and have been implicated in the mechanisms of tumour invasion and metastatic spread. We have used immunohistochemistry to examine and compare the tissue distributions of collagenase-1 (MMP-1), gelatinase A (MMP-2) and stromelysin-1 (MMP-3) in 18 specimens of malignant melanoma, viz. 10 superficial spreading and 8 nodular melanomas. MMPs-1, -2 and -3 were demonstrated within melanoma and host tissue cells, especially at the periphery of some tumours, but were usually restricted to less than 10% of total melanoma cells. The MMPs were absent from 'normal' skin tissue distant from the tumour. MMP-2 was localised to discrete groups of cells and was especially evident at the epidermal:tumour interface, whereas MMP-3 was mainly confined to the deeper margins of melanoma. No regular pattern of MMP expression was observed for either the superficial spreading or the nodular melanomas. The variable distributions of the MMPs suggested that enzyme expression was subject to local microenvironmental regulation, possibly in response to matrix components and the cellular heterogeneity observed at the tumour margins. These in situ observations add weight to the concept that specific MMPs contribute to the mechanisms of tumour invasion.

Evidence for in vivo mitosis by granule-containing mast cells from canine mastocytomas.

Mast cell accumulations are generally considered to arise almost exclusively from the recruitment of non-granulated, bone-marrow-derived, precursor cells, with the stem cell factor (SCF) reported to play a crucial role in the growth, development and maturation of granulated mast cells within specific tissue sites. In this study dog mastocytoma specimens have been examined by both immunohistochemical and ultrastructural techniques, to demonstrate that fully granulated mast cells are capable of mitotic activity. Observations showing the formation of mitotic spindles, chromosome separation and cytokinesis all support the concept that granulated mast cells are capable of proliferative activity. The ability of mature granulated mast cells to replicate provides an alternative process for local increases in mast cell numbers, at least in canine mast cell tumours. Such observations suggest the possibility that normal or neoplastic human mast cells, fully granulated, have the potential to proliferate in specific tissue sites.

Comparative studies of collagenase and stromelysin-1 expression by rheumatoid synoviocytes in vitro.

Matrix metalloproteinases such as collagenase and stromelysin are recognised as important cartilage-degrading enzymes in the pathophysiology of rheumatoid arthritis. Synovial fibroblasts and macrophages are the major cellular components of rheumatoid synovium, but the regulation and relative expression of collagenase and stromelysin by these two cell types remains uncertain. Using in vitro cultures of adherent rheumatoid synovial cells we have examined the coordinate or separate expression of collagenase and stromelysin-1 by dual immunolocalisation and Western blotting techniques. Synovial fibroblasts, when activated by macrophage-derived products in primary culture or by interleukin-1/phorbol myristate acetate in subcultures, released significant quantities of collagenase and stromelysin in their inactive, precursor forms. The ratio of released procollagenase: prostromelysin varied between different synovial cell preparations. Dual immunolocalisation studies demonstrated both coordinate and separate expression of the two enzymes by single cells. Approximately 80% of the activated fibroblasts, especially those with stellate morphology, showed co-expression of both enzymes. By contrast synovial macrophages had a modest or negligible capacity to elaborate either enzyme under the same in vitro conditions. In many fibroblastic cells both collagenase and stromelysin were co-localised to the perinuclear Golgi region and the same cytoplasmic compartments. Vesicular structures appear to provide intracellular transport for both enzymes to sites of secretion. Both enzymes showed preferential pericellular binding to a collagenous substratum rather than any association with the plasma membrane/cell surface.

Clinical, biochemical, and immunohistochemical features of necrobiotic xanthogranulomatosis.

AIMS: To describe the clinical features of two patients with paraproteinaemia and necrobiotic xanthogranulomatosis together with detailed immunohistochemistry of the lesions in one. METHODS: The clinical history and results of biochemical investigations of the patients were retrieved from the files. Immunohistochemistry was used to investigate the expression of macrophage and mast cell markers, amyloid A and P, S-100 protein, and apolipoprotein AI and B in xanthogranulomatous skin lesions from patient 2. In addition, protein A-sepharose chromatography was used to separate serum from patient 2 and apolipoprotein B and the IgG paraprotein were measured in the fractions eluted. RESULTS: Monocytes/macrophages comprised the major cellular component of the lesion, and unusually for xanthomata, areas of collagen necrosis were also seen. Activated mast cells were present at the margins of macrophage clusters and adjacent to areas of collagen necrosis. Serum paraprotein was bound to low density lipoproteins as judged by protein A-sepharose chromatography, and was also located within macrophagic foam cells of the lesion on immunohistochemistry. CONCLUSIONS: These observations demonstrate many features similar to atherosclerosis including collagen necrosis and mast cell activation.

The short-acting anesthetic propofol produces biphasic effects-depression and withdrawal rebound overshoot-on some (but not all) limbic evoked potentials in the behaving rat.

Propofol, the relatively new, short-acting general anesthetic, markedly enhances the action of GABA at the GABAA receptor. To evaluate its effects on field potentials evoked in the dentate gyrus (DG) during the anesthetic and recovery periods, propofol was administered intraperitoneally to behaving rats bearing stimulating electrodes in the dorsal perforant path (DPP), where medial perforant path fibers predominate, and in the anterior piriform cortex (PC; i.e., olfactory cortex), and recording electrodes in the DG. Input from the PC reaches the DG via the lateral perforant path. Population slow waves (SWs) were evoked by paired-pulse stimulation of the PC at a 32 ms interstimulus interval (ISI) to produce paired-pulse facilitation in the awake animal. We had previously demonstrated that amplitude of SW2 (produced by the second stimulus) was greatly decreased by GABAergic drugs and increased by antiGABAergic convulsant agents. After administration of propofol, mean amplitude of SW2 decreased immediately and remained low for 30-60 min during propofol-induced sleep (as expected), then unexpectedly increased to about 1.5- to 2-fold above pretreatment levels at 2-4 h before gradually returning to pretreatment levels. In addition, the DPP was stimulated to produce either paired-pulse inhibition (20 ms ISI) or facilitation (32 ms ISI) of DG population spikes (PSs) in the awake animal. PS2 was much more inhibited during propofol-induced sleep, than during the pretreatment period, consistent with an expected marked increase in recurrent inhibition. An overshoot in PS2 amplitude was observed only occasionally during recovery, suggesting that withdrawal overshoot in amplitudes is more characteristic of PC-evoked DG SW2 potentials. The overshoot in SW2 amplitude during recovery may have been related to propofol's 'rapid on-rapid off' actions on the GABAA receptor, perhaps resulting in a phenomenon like the 'GABA withdrawal syndrome'. Such an effect, if true, may help explain the rare occurrence of seizures, especially during recovery, associated with its use clinically.

Morphometric effects of postnatal lead exposure on hippocampal development of the 15-day-old rat.

Neurotoxic sequelae of developmental lead exposure suggest that the hippocampus may be affected. Therefore, rats received low-level exposure via the milk of dams drinking 0.2% lead acetate beginning at parturition, and mid-dorsal sections of the hippocampus and dentate gyrus (DG) from 15-day-old pups were examined by light and electron microscopy. Lead exposure did not reduce body weight nor produce obviously abnormal vascularity or signs of cytotoxicity in the hippocampal formation, and total numbers per section of dentate granule cells or CA3 pyramidal cells were not reduced. On the other hand, lead exposure reduced neuropil development as evidenced both by reduced areas of the dentate hilus and dentate infrapyramidal stratum moleculare and by increased number of hilar CA3 pyramidal cells per unit area. Also, lead exposure reduced numbers of several types of synaptic profiles per unit area in the suprapyramidal mossy fiber zone. Complex invaginated (CI) profiles, assumed to be mature mossy fiber boutons, were characterized by multiple membrane densities and deep invaginations around dendritic spines of pyramidal cells. Complex noninvaginated (CN) boutons exhibited bag-like profiles with multiple membrane densities. Smaller, less numerous, simple (S) profiles contacted either dendritic trunks (ST) or spines (SS). Lead exposure reduced the numerical density of any of the profiles in the deep (close to stratum pyramidale) part of the proximal (close to DG) region of the suprapyramidal mossy fiber zone, but did not alter the numerical density of any of the profiles in the superficial (distal to stratum pyramidale) parts of either proximal or distal (close to CA1) regions. Average size of CN profiles in the distal region was increased by lead exposure. The pattern of effects suggests that low-level lead exposure during development preferentially affects later developing structures within the hippocampal formation, rather than affecting mature structures.

Ro 5-4864, like picrotoxin, enhances EPSP-spike coupling in the freely behaving rat.

To help determine its mechanism of action, the convulsant benzodiazepine Ro 5-4864 was administered (15-20 mg/kg) intraperitoneally (IP) and electrophysiological and behavioral effects were compared; parallel studies were conducted with picrotoxin (PTX; 1 mg/kg). Both PTX and Ro 5-4864 produced myoclonic seizures, primarily between 15-40 min after administration; myoclonus was followed by more severe seizures after PTX. Both Ro 5-4864 and PTX produced a maximal increase in amplitude and decrease in threshold of the population spike (PS) evoked in the dentate gyrus (DG) by stimulation of the dorsal perforant path prior to peak seizure activity; start latency of the PS and initial slope and amplitude of the population slow wave (SW) were not changed. Amplitude of the PS was already increased by 5 min after administration of Ro 5-4864 and was maximally increased 1.8- to 3-fold, depending on stimulus intensity, usually by 10 min. Similarly, by 20 min after administration, PTX had also increased PS amplitude in the absence of an effect on PS latency or the SW. The increase in PS amplitude without concomitant changes in the SW suggests that Ro 5-4864 enhanced coupling between the excitatory postsynaptic potential (EPSP) and firing of the postsynaptic neurons, i.e., it enhanced E-S coupling, as has also been suggested for PTX. The similarity in the effects of Ro 5-4864 and PTX suggests that antiGABAergic effects, perhaps along feedforward inhibitory pathways, are involved in both the seizures and enhanced E-S coupling.

Hippocampal potentials evoked by stimulation of olfactory basal forebrain and lateral septum in the rat.

Electrophysiological characteristics of olfactory-hippocampal relations were examined because recent anatomical studies have described a substantial olfactory input to the hippocampus via the entorhinal cortex. Potentials evoked in the dorsal hippocampus of anesthetized rats by stimulation of the prepyriform cortex, pyriform cortex, diagonal band, lateral olfactory tract, anterior commissure, olfactory tubercle and anterior olfactory nucleus had similar characteristics, although latencies differed. For example, latencies were twice as long after stimulation of the obliquely oriented portion of the diagonal band than after stimulation of the prepyriform cortex. A relatively low-amplitude, initially negative wave was recorded in the subiculum, CA1 and CA2, and a relatively high-amplitude, initially positive wave was recorded in CA4 and the dentate gyrus. In CA3 negative potentials were observed at dorsal recording sites and positive potentials were recorded at more ventral sites. Peak latencies were usually two to four msec shorter for the negative than for the positive wave. Laminar distributions of responses evoked in the hippocampus by stimulation of the prepyriform cortex and diagonal band were evaluated by driving eight electrodes mounted on one carrier through the brain and were found to be strikingly similar. Maximal amplitudes of the negative wave were recorded at the level of stratum moleculare of CA1 and the subiculum, and peak amplitudes of the positive wave were associated with the hilus of the dentae gyrus. Transition from negative to positive waveforms occurred approximately at the hippocampal fissure. Although the negative and positive waves were usually elicited together, they also were separable in that only negative waves were recorded along some tracks and only positive waves along others. Also, various stimulation sites in the prepyriform cortex elicited stable high-amplitude positive waves accompanied by negative waves of varying amplitude. It is suggested that branches of the perforant path are involved in generation of the two waves and that activity in a number of olfactory structures may influence the hippocampus, probably via the perforant pathway. Thus, hippocampal potentials following prepyriform or diagnonal band stimulation were not abolished by transection of the fornix-fimbria. Dorsolateral septal stimulation evoked hippocampal responses with characteristics and distribution distinctly different from those evoked by stimulation of olfactory areas. The findings suggest that lateral septal stimulation may activate the hippocampus antidromically.

A perspective of lead poisoning in antiquity and the present.

Seen in perspective, it is evident that lead poisoning is one of the earliest occupational diseases, described already thousands of years ago. The first major upswing in the history of anthropogenic production of lead was associated with the development of the Greco-Roman culture and the most recent followed the Industrial Revolution. At the peak of the power of the Roman Empire, lead production was about 80,000 tons per year, lead and its compounds were used with great inventiveness in numerous ways, and lead poisoning was pandemic, with the severity of poisoning proportional to the power and status of the class. Intake of lead by the aristocracy may have been as much as 1 mg/day. The resultant mental incompetence and especially the rapidly declining birth rate among the ruling class are now believed to have been major factors in the decline of the Roman Empire. Epidemic outbreaks of lead poisoning have occurred repeatedly throughout history and still occur today. The estimated 3.5 million tons of lead produced annually during peak production in the 1970's included about 0.4 million tons of organoleads. Such intense production has increased global contamination with lead enormously. Even under relatively ideal conditions the daily intake of lead is so much higher than in prehistoric times that investigators must pause to ask themselves what a proper control group really is. Are investigators merely determining the effects of additional lead exposure on systems already greatly perturbed by lead? If so, how can we find out?