Ultrastructural quantitation of atubular and hypertrophic glomeruli in rats with lithium-induced chronic nephropathy

Summary The very heterogeneous population of glomeruli in rats with lithium-induced chronic nephropathy which includes small glomeruli without connection to a proximal tubule (atubular glomeruli) and large hypertropic glomeruli with connection to a normal proximal tubule, was studied at the ultrastructural level, using stereological methods. After 8 weeks of lithium treatment followed by 8 weeks without lithium the hypertrophic glomeruli showed no changes in their relative ultrastructural composition, including normal mesangium, basement membrane-like material and peripheral basement membrane. The absolute quantities of each component were, however, increased due to the increased volume of the glomeruli. The atubular glomeruli had increased volume fractions of mesangium, peripheral basement membrane, basement membrane-like material and epithelium, whereas the absolute quantities were decreased due to the decreased volume. The thickness of the basement membrane was within normal limits in the group of hypertrophic glomeruli but increased by 31% above controls in the group of atubular glomeruli. Both groups of glomeruli in lithium-treated animals showed normal mean foot process width, but with a slightly abnormal distribution. The atubular glomeruli showed a disproportionate large decrease in peripheral filtration surface and capillary length, compared with the reduction in glomerular volume, whereas the hypertrophic glomeruli showed changes in proportion with the increased volume.     

Hepatocellular Carcinoma: An Update

Hepatocellular carcinoma (HCC) is the most common malignant tumor of males in the world, with an incidence of 1,000,000 new cases a year. It is endemic in Southeast Asia and Sub-Saharan Africa. Risk factors include chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), Aflatoxin B1 uptake, hemochromatosis, and &#102 1-antitripsin deficiency. Epidemiological studies provide evidence for the association of HCC with HBV infection. The incidence of HCC is high in regions hyperendemic for HBV. Chronic carrier state and maternal-infant transmission are important factors in the development of HCC. Evidence of direct oncogenic effect of HBV is well established, HCCs contain viral DNA sequences integrated into hepatocyte DNA that act as random insertional mutagens, and these sites are near genes involved in the control of proliferation and differentiation. The mechanism of hepatitis C virus in hepatocarcinogenesis is still imprecise but a high percentage of cases are related to this virus. Chronic alcohol consumption and cirrhosis are cofactors that increase the development of HCC in patients with chronic viral infection. In experimental carcinogenesis a multipotential element called oval cell proliferates in the early stages. The cellular events are accompanied by increased expression of several growth factors that enhance the survival of carcinogen-activated cells by suppressing apoptosis and increasing elements entering the cell cycle. Hepatic carcinogenesis is a complex process associated with accumulation of genetic and epigenetic changes that run through steps of initiation, promotion and progression. Activation of oncogenes of the "ras" family and others has been detected during chemically-induced HCC in rodents, but there is little evidence of such activation in human tumors. The role of tumor supressor genes such as retinoblastoma (RB) and P53 genes has been documented. Aflatoxin B1 that contaminates foods in endemic areas has a clear role in hepatocarcinogenesis. Metabolites of this toxin promote apurinic sites and G to T mutations in chromosomal DNA, the third base of codon 249 of the P53 gene is preferentially targeted to form aducts with aflatoxin B1, and this mutation has been specifically identified in HBV infection. Histological and cytological criteria for the diagnosis of HCC are well established and are based in architectural and cytological changes. An important issue is the diagnosis of liver "nodules" detected by image, from which small biopsies or aspiration material is obtained. Special studies such as reticulin, CD34, cytokeratin profile, and MOC-31 can be very useful for the differential diagnosis of primary and metastatic tumors. Telomerase activity has been found in HCC and negative in pericancerous tissue. It is more pronounced in poorly differentiated tumors and correlates with factors of clinical importance, such as prognosis and recurrences. Cells of well-differentiated HCC have an ultrastructural appearance similar to normal hepatocytes. During the process of dedifferentiation, there is progressive loss of organization of intracellular organelles. The cell cohesion is lost, intercellular gaps with microvilli appear, the sinusoids become capillarized, and reparative changes are seen in the spaces of Disse. A variety of inclusions, such as Mallory bodies, granular material, secondary lysosomes, and Dubin-Johnson pigment, have been described. Fibrolamellar carcinoma has a characteristic histological picture and ultrastructurally oncocytic features. Neuroendocrine granules and combination of HCC with bile duct carcinoma are seen by electron microscopy.     

Binding and degradation of 125I-insulin by renal glomeruli and tubules isolated from rats

Summary Isolated rat renal glomeruli and tubules were shown to exhibit specific binding of ¹²⁵I-insulin and enzymatic degradation of the hormone. Binding to both renal fractions reached a plateau by 1h at 22 °C and increased linearly with increasing protein concentrations. Binding was inhibited in both preparations by insulin and its analogues in the order of relative potency: insulin > despentapeptide insulin > proinsulin, but insulin was ten times more potent in inhibiting ¹²⁵I-insulin binding to glomeruli than that to tubules, indicating a different affinity of receptors for the hormone in the two renal fractions (about 17 versus 210 g unlabelled insulin/l inhibiting 50% of the ¹²⁵I-insulin binding to glomeruli and tubules, respectively). Bound ¹²⁵I-insulin dissociated at a faster rate from tubules than from glomeruli; this release was accelerated by unlabelled insulin in both renal fractions, but to a greater extent in glomeruli than in tubules. Two-thirds of the total bound material released from glomeruli was found to be intact insulin as measured by trichloroacetic acid precipitation, whereas only one-third of the material released from tubules was intact. No direct relationship between binding and degradation of ¹²⁵I-insulin in these renal fractions could be demonstrated, however, because of the release of proteolytic enzymes into the incubation medium resulting in almost all degradation being extracellular. Although differing in their affinity for ¹²⁵I-insulin the high affinity glomerular insulin receptor and the lower affinity tubular insulin receptor have characteristics similar to those of insulin receptors in insulin responsive tissues.     

From the Cover: Intergenerational transmission of emotional trauma through amygdala-dependent mother-to-infant transfer of specific fear

Emotional trauma is transmitted across generations. For example, children witnessing their parent expressing fear to specific sounds or images begin to express fear to those cues. Within normal range, this is adaptive, although pathological fear, such as occurs in posttraumatic stress disorder or specific phobias, is also socially transmitted to children and is thus of clinical concern. Here, using a rodent model, we report a mother-to-infant transfer of fear to a novel peppermint odor, which is dependent on the mother expressing fear to that smell in pups’ presence. Examination of pups’ neural activity using c-Fos early gene expression and ¹⁴C 2-deoxyglucose autoradiography during mother-to-infant fear transmission revealed lateral and basal amygdala nuclei activity, with a causal role highlighted by pharmacological inactivation of pups’ amygdala preventing the fear transmission. Maternal presence was not needed for fear transmission, because an elevation of pups’ corticosterone induced by the odor of the frightened mother along with a novel peppermint odor was sufficient to produce pups’ subsequent aversion to that odor. Disruption of axonal tracts from the Grueneberg ganglion, a structure implicated in alarm chemosignaling, or blockade of pups’ alarm odor-induced corticosterone increase prevented transfer of fear. These memories are acquired at younger ages compared with amygdala-dependent odor-shock conditioning and are more enduring following minimal conditioning. Our results provide clues to understanding transmission of specific fears across generations and its dependence upon maternal induction of pups’ stress response paired with the cue to induce amygdala-dependent learning plasticity. Results are discussed within the context of caregiver emotional responses and adaptive vs. pathological fears social transmission.Children, including infants, use their parents’ emotions to guide their behavior and learn about safety and danger (1–4). The infant’s ability to regulate behavior in novel situations using the caregiver’s emotional expression is known as social referencing and occurs in humans and nonhuman primates (1). Although parental physical presence itself or particular cues indicating parental presence, such as voice, touch, or smell typically signal safety for the child, infants are especially responsive to the caregiver’s communication during threats (3–5). This social learning is critical for enhancing survival through an adaptation to the environment but also provides transmission of pathological fears, such as occurs in posttraumatic stress disorder (PTSD) or in specific phobias (3–7).Despite existing evidence that children are sensitive to parental fear and anxiety, the neurobiological mechanisms for the transmission of parental specific fear to the offspring have remained elusive (2–7). Animal studies investigating the impact of parental stress on the offspring focused on the history of parental trauma, quality of maternal care, and resultant overall behavioral alterations in the offspring (7, 8). However, to develop efficient survival strategies, progenies must learn about specific environmental threats triggering parental fear (9).Most of what we know about fear learning comes from studies using fear conditioning (FC) (10). In FC, a neutral sensory cue [conditioned stimulus (CS)] is paired with a noxious event [unconditioned stimulus (US)]. Animal studies indicate that the amygdala’s lateral and basal nuclei (LBA) play an important role in FC (10). However, FC in infant rats is naturally attenuated until postnatal day (PND) 10 due to low levels of the stress hormone corticosterone (CORT) during the stress hyporesponsive period (11–15). This fear suppression continues in older pups (until PND 16) in the mother’s presence due to social buffering (attenuation) of the shock-induced CORT increase (15).To study the intergenerational transmission of fear to specific triggers, we developed a mother-to-infant social fear learning paradigm. In social fear learning, an organism learns fear through an exposure to a conspecific expressing fear to a discrete CS. Social fear learning may thus serve as a model explaining how defense responses to specific triggers are transmitted between individuals. Social fear learning has been demonstrated in primates, including humans and in rodents, and involves the amygdala (16–19).     

Developmental profiles of various cholinergic markers in the rat main olfactory bulb using quantitative autoradiography

The existence of possible relationships among the developmental profile of various cholinergic markers in the main olfactory bulb (OB) was assessed by using in vitro quantitative autoradiography. Muscarinic receptors were visualized with [³H]pirenzepine (muscarinic M1-like sites) and [³H]AF-DX 384 (muscarinic M2-like sites); nicotinic receptors by using [³H]cytisine (nicotinic 42-like subtype) and [¹²⁵I]α-bungarotoxin (nicotinic 7-like subtype); cholinergic nerve terminals by using [³H]vesamicol (vesicular acetylcholine transport sites) and [³H]hemicholinium-3 (high-affinity choline uptake sites). These various cholinergic markers exhibited their lowest levels at birth and reached adult values by the end of the 4–5 postnatal weeks. However, the density of presynaptic cholinergic markers and nicotinic receptors at postnatal day 2 represented a large proportion of the levels observed in adulthood, and displays a transient overexpression around postnatal day 20. In contrast, the postnatal development of cholinergic muscarinic M1-like and M2-like receptors is apparently regulated independently of the presynaptic cholinergic markers and nicotinic receptors. Two neurochemically and anatomically separate olfactory glomeruli subsets were observed in the posterior OB of the developing rat. These atypical glomeruli expressed large amounts of [³H]vesamicol- and [³H]hemicholinium binding sites without significant amounts of muscarinic M1, M2, or nicotinic α4β2 receptor binding sites. A significant density of [¹²⁵I]α-bungarotoxin binding sites could be detected only at early postnatal ages. A few olfactory glomeruli specifically restricted to the dorsal posterior OB expressed a high density of [³H]cytisine binding sites but lacked significant binding of the two presynaptic cholinergic markers used here, suggesting their noncholinergic but cholinoceptive nature. © 1996 Wiley-Liss, Inc.     

Cell surface carbohydrates and glomerular targeting of olfactory sensory neuron axons in the mouse

Cell surface carbohydrates have been implicated in axon guidance and targeting throughout the nervous system. We have begun to test the hypothesis that, in the olfactory system, a differential distribution of cell surface carbohydrates may influence olfactory sensory neuron (OSN) axon targeting. Specifically, we have examined the spatial distribution of two different plant lectins, Ulex europaeus agglutinin (UEA) and Dolichos biflorus agglutinin (DBA), to determine whether they exhibit differential and reproducible projections onto the main olfactory bulb. Each lectin exhibited a unique spatial domain of glomerular labeling that was consistent across animals. UEA labeling was strongest in the ventral aspect of the olfactory bulb; DBA labeling was strongest in the dorsal aspect of the olfactory bulb. Some evidence for colocalization was present where these two borders intersected. Large areas of the glomerular layer were not labeled by either lectin. To determine whether patterns of lectin labeling were reproducible at the level of individual glomeruli, UEA labeling was assessed relative to M72-IRES-taulacZ- and P2-IRES-taulacZ-labeled axons. Although glomeruli neighboring these two identified glomeruli were consistently labeled with UEA, none of the lacZ positive axons was lectin labeled. Labeling of vomeronasal sensory neuron axons in the accessory olfactory bulb was more uniform for the two lectins. These data are the first to show a differential distribution of UEA vs. DBA labeling in the main olfactory bulb and are consistent with the hypothesis that a differential distribution of cell surface carbohydrates, a glycocode, may contribute to the targeting of OSN axons.     

Radial glia development in the mouse olfactory bulb

Radial glia are critical for cell migration and lamination of the cortex. In most developing cortical structures, radial glia, as their name suggests, extend processes from the ventricle to the pia in regular parallel arrangements. However, immunohistochemical labeling from several laboratories suggests that radial glia have a more branched morphology in the olfactory bulb. To investigate the morphology of radial glia in the mouse olfactory bulb we (1) labeled radial glia and olfactory receptor neuron axons at 24-hour intervals by immunohistochemistry; and (2) developed a novel method of generating and applying "nanocrystals" of 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) to the ventricle surface such that the processes of single olfactory bulb radial glia are labeled in the embryonic olfactory bulb. We examined the structure and interactions of radial glia with ingrowing olfactory receptor neuron (ORN) axons in late embryonic olfactory bulb development. These results showed that olfactory bulb radial glia do not form straight parallel structures as do radial glia in the neocortex but rather have a convoluted trajectory from the ventricle to the bulb surface. Moreover, olfactory bulb radial glia consistently extend tangential branches at the level of the internal plexiform layer. Beginning at embryonic day 17.5, two types of radial glia can be distinguished: type I radial glia have a process that extends from the ventricle into the glomerular layer. These apical processes form highly restricted tufts, or "glial glomeruli" at the same time that ORN axons are forming "axonal glomeruli." In type II radial glia the apical process does not enter the glomerular layer but instead ramifies within the external plexiform layer. The tight spatiotemporal relationship between the glomerulization of radial glia processes and ORN axons during development suggest that radial glia processes could play a role in the formation and/or stabilization of mammalian glomeruli.     

Characterization of an antiserum to synaptic glomeruli from rat cerebellum

Rabbit anti-rat cerebellar synaptic glomeruli antiserum when absorbed with non-neural tissues reacts only with neural tissues when tested by indirect immunofluorescence on tissue sections. Further absorption with forebrain results in an antiserum which detectably reacts only with synaptic glomeruli and soma of Purkinje cells of both rat and mouse. The developmental expression of the synaptic glomeruli antigen(s) parallels the formation of synapses between mossy fibers and granule cells. Immature synaptic contacts do not contain recognizable antigent(s), whereas only at postnatal Day 15 glomeruli become antigen-positive. At this stage antigen in Purkinje cells is no longer carried in their dendrites, but becomes confined to the cell soma. Staggerer mutant mice still express the immature pattern of antigen distribution on postnatal Day 18.     

Differential activation of glomeruli in the ferret's main olfactory bulb by anal scent gland odours from males and females: an early step in mate identification

Peripheral anosmia was previously found to disrupt sex discrimination and partner preference in male and female ferrets. Here we show directly that volatile anal scent gland odourants from male and female ferrets activated overlapping but distinguishable clusters of glomeruli located in the ventral-caudal portion of the main olfactory bulb (MOB) of breeding ferrets of both sexes. No glomerular activation was seen in the accessory olfactory bulb (AOB). The profile of MOB glomerular activation induced in oestrous females by male anal scents was very similar to that induced by direct contact with a male during mating, and oestrogen treatment failed to alter the profile of glomerular activation induced in ovo-hysterectomized females by male anal scents. In rodents, 'atypical' MOB glomeruli, which have dense acetylcholinesterase (AChE) activity in the neuropil, may be activated by body odours from conspecifics. No such AChE-staining 'atypical' glomeruli were found in the ferret's MOB, suggesting that in this carnivore they do not constitute a subset of MOB glomeruli that respond to body odourants. In ferrets of both sexes, volatile body odourants that are detected by the main as opposed to the vomeronasal-AOB accessory olfactory system may play a critical role in mate identification.