Atypical Glomeruli Research Articles

Spatial Heterogeneity of Glomerular Phenotypes Affects Kidney Biopsy Findings.

Detection of rare glomerular phenotypes can impact diagnosis in indication kidney biopsies and in kidney tissue used for research studies. Nephropathologists are aware of potential sampling error when assessing needle biopsy cores but quantitative data are lacking. Kidney tissue from patients undergoing total nephrectomy enrolled in an observational, cross-sectional cohort study was used to characterized glomeruli as typical or atypical, which included globally sclerosed glomeruli (GSG), segmentally sclerosed glomeruli (SSG), ischemic-like, and imploding. A 2D map of the glomerular annotations was generated. Spatial centrality of atypical glomeruli using the L2 metric and differences in pairwise distances between typical or atypical glomeruli were calculated. To determine how the yield of capturing atypical glomerular phenotype was affected by biopsy depth (i.e. not including the renal capsule), simulated kidney biopsies were generated from the 2D map. The mean number of glomeruli in a nephrectomy specimen was 209 (SD 143), and GSG were the most common type of atypical glomeruli (median: 13% [IQR: 5,31]). Typical glomeruli were more likely to be surrounded by other glomeruli (i.e. centrally located in kidney cortex) than GSG, SSG, ischemic-like and imploding glomeruli. Atypical glomeruli were 7.3% (95% CI 4.1, 10.4) closer together than typical glomeruli and were more likely to be closer together in older patients or those with hypertension. In simulated kidney biopsies, failure to capture the capsule was associated with under-detection of GSG, ischemic-like and imploding glomeruli. Spatial analysis of large sections of kidney tissue provided quantitative evidence of spatial heterogeneity of glomerular phenotypes including clustering of atypical glomeruli in individuals with hypertension or older age. Most importantly, deep kidney biopsies that lack subcapsular area under-detect atypical glomerular phenotypes, suggesting that capturing the renal capsule as an important quality control measure for kidney biopsies.

The effects of dexmedetomidine on early acute kidney injury in severely burned rats.

Burns are a global medical and economic problem. In addition to high costs, the lengthy therapeutic process and the emotional trauma experienced by patients and their families indirectly worsen the socioeconomic damage caused. Kidney failure observed after burns is highly correlated with mortality. Twenty-eight male Sprague-Dawley rats (age four months, weight 250-350 g) were included in the study. They were randomly assigned into four groups consisting of seven rats each with similar mean weights. Group 1 (n=7) represented the healthy control group (C), Group 2 (n=7) the Sham+dexmedetomidine (DEX) 100 mcg/kg (three doses) (S+DEX100) group, Group 3 (n=7) the 30% Burn (B), and Group 4 (n=7) the 30% Burn+DEX 100 mcg/kg/day group (B+DEX100) (three doses). Thiobarbituric acid reactive substances (TBARS), total thiol (TT), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) values in kidney tissues were investigated biochemically, and histopathological analyses were also performed. Nuclear factor κB (NF-κB)/p65 was measured using immunohistochemistry, and the TUNEL assay was applied to indicate apoptotic tubular epithelial cells. TBARS, IL-1, and TNF-α in kidney tissues decreased in the B+DEX100 group compared to the 30% burn group, while total thiol values increased. Histopathologically, atypical glomeruli, particularly necrotic tubules, and inflammation in peritubular areas decreased in the B+DEX100 group compared to the 30% burn group. In addition, apoptotic tubular epithelial cells exhibiting TUNEL positivity and tubular epithelial cells exhibiting NF-кβ/p65 positivity also decreased in the B+DEX100 group compared to the 30% burn group. Dexmedetomidine reduced apoptotic activity in rats and exhibited anti-inflammatory antioxidant effects in the burn model in this study.

Periodontitis exacerbates the renal degenerative effects of obesity in rats.

Obesity and periodontitis are systemic subclinical inflammatory diseases with established negative renal effects. The aim of this animal study was to thoroughly investigate the possible effects of these two diseases on renal structure and function. Thirty-two male Sprague Dawley rats were divided into four groups: control (C), obesity (Ob), experimental periodontitis (Ep), and Ob+Ep. The first 16weeks of the experiment were aimed for the induction of obesity and the last 5weeks for the induction of periodontitis. Throughout the experimental period, the C and Ep groups were fed standard rat chow, while the Ob groups (Ob and Ob+Ep) were fed high-fat rat chow. Right after the establishment of obesity, periodontal tissue destruction was achieved by placing 3.0silk sutures in sub-paramarginal position around the cervices of mandibular right-left first molar teeth and preserving them for 5weeks. On the last day of the 22nd week, following blood collection, all rats were euthanized, and kidneys and mandibles were collected. Alveolar bone loss was measured on microcomputed tomographic slices. Histopathological evaluations (light microscopy, semi-quantitative analysis of renal corpuscle area, and immunohistochemical analysis of caspase-3 activity) were done on right kidneys and biochemical evaluations (malonyl-aldehyde [MDA], glutathione [GSH], total oxidant status [TOS], total antioxidant status [TAS], oxidative stress [OSI], tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], matrix metalloproteinase [MMP]-8, MMP-9, and cathepsin D [CtD] levels) were done on left kidneys. Renal functional status was evaluated with levels of serum creatinine, urea, and cystatin C. Periodontal bone loss was significantly higher in the Ep and Ob+Ep groups, compared with the C and Ob groups (p<.05). All parameters except TAS and GSH were highest in the Ob+Ep group, and the differences were statistically significant compared with the control group (p<.05). Although the mean TAS and GSH levels were lower in the Ob+Ep group than the other groups, the differences were not statistically significant (p>.05). While the atypical glomeruli score was significantly higher in the Ob+Ep group than in all other groups (p<.05), the acute tubular necrosis and histopathological scores were significantly different only compared with the control group (p<.05). This experimental study showed that the negative effects of the co-existence of periodontitis and obesity on inflammatory stress and apoptotic changes in the kidneys together with the functional parameters were significantly more severe, compared with the presence of one of these diseases alone. TNF-α could have a central role in the periodontitis and obesity-related structural and functional renal changes.

Shaping of the nephron \u2013 a complex, vulnerable, and poorly explored backdrop for noxae impairing nephrogenesis in the fetal human kidney

BackgroundThe impairment of nephrogenesis is caused by noxae, all of which are significantly different in molecular composition. These can cause an early termination of nephron development in preterm and low birth weight babies resulting in oligonephropathy. For the fetal human kidney, there was no negative effect reported on the early stages of nephron anlage such as the niche, pretubular aggregate, renal vesicle, or comma-shaped body. In contrast, pathological alterations were identified on subsequently developing S-shaped bodies and glomeruli. While the atypical glomeruli were closely analyzed, the S-shaped bodies and the pre-stages received little attention even though passing the process of nephron shaping. Since micrographs and an explanation about this substantial developmental period were missing, the shaping of the nephron in the fetal human kidney during the phase of late gestation was recorded from a microanatomical point of view.ResultsThe nephron shaping starts with the primitive renal vesicle, which is still part of the pretubular aggregate at this point. Then, during extension of the renal vesicle, a complex separation is observed. The medial part of its distal pole is fixed on the collecting duct ampulla, while the lateral part remains connected with the pretubular aggregate via a progenitor cell strand. A final separation occurs, when the extended renal vesicle develops into the comma-shaped body. Henceforth, internal epithelial folding generates the tubule and glomerulus anlagen. Arising clefts at the medial and lateral aspect indicate an asymmetrical expansion of the S-shaped body. This leads to development of the glomerulus at the proximal pole, whereas in the center and at the distal pole, it results in elongation of the tubule segments.ConclusionsThe present investigation deals with the shaping of the nephron in the fetal human kidney. In this important developmental phase, the positioning, orientation, and folding of the nephron occur. The demonstration of previously unknown morphological details supports the search for traces left by the impairment of nephrogenesis, enables to refine the assessment in molecular pathology, and provides input for the design of therapeutic concepts prolonging nephrogenesis.

Action Plan for Prolongation of Nephrogenesis in Preterm and Growth Restricted Babies: Explore Ultrastructure of the Nephrogenic Zone, Identify a Molecular Target, Select a Viable Drug and Find a Path for Administration.

A large amount of investigations informs about primary steps of mammalian kidney development such as anlage of the organ and initial nephron formation, while only few data exists about the late phase of human kidney development. In particular, little attention was up to date addressed to the decrease of morphogenic activity in the nephrogenic zone short before birth and the vanishing of all stem cell niches aligned beyond the organ capsule. There is evidence that molecular controlling of this normal but degenerative developmental process also plays a decisive role in the kidneys of preterm and growth restricted babies. Although they are born in a phase of active nephrogenesis, a substantial percentage of them evolves oligonephropathy, formation of atypical glomeruli and immaturity of parenchyma. Pathologic findings point out that independent from chemical nature all suspected hampering influences sublimate in the nephrogenic zone. However, it is unknown, whether impaired nephrogenesis is locally caused by harming interstitial fluid, disturbance of morphogen signaling, unbalanced synthesis of extracellular matrix or limited cell to cell communication. Thus, first of all these issues must be resolved, then save application of medicines prolonging nephrogenesis waits for realization. Due to the unexpectedly complex microanatomy and physiology of the nephrogenic zone, it will be a particular challenge for the future.

Differential effects of unilateral olfactory deprivation on noradrenergic and cholinergic systems in the main olfactory bulb of the rat

The lack of environmental olfactory stimulation produced by sensory deprivation causes significant changes in the deprived olfactory bulb. Olfactory transmission in the main olfactory bulb (MOB) is strongly modulated by centrifugal systems. The present report examines the effects of unilateral deprivation on the noradrenergic and cholinergic centrifugal systems innervating the MOB. The morphology, distribution, and density of positive axons were studied in the MOBs of control and deprived rats, using dopamine-β-hydroxylase (DBH)-immunohistochemistry and acetylcholinesterase (AChE) histochemistry in serial sections. Catecholamine content was compared among the different groups of MOBs (control, contralateral, and ipsilateral to the deprivation) using high-performance liquid chromatography analysis. Sensory deprivation revealed that the noradrenergic system developed adaptive plastic changes after olfactory deprivation, including important modifications in its fiber density and distribution, while no differences in cholinergic innervation were observed under the same conditions. The noradrenergic system underwent an important alteration in the glomerular layer, in which some glomeruli showed a dense noradrenergic innervation that was not detected in control animals. The DBH-positive glomeruli with the highest noradrenergic fiber density were compared with AChE-stained sections and it was observed that the strongly noradrenergic-innervated glomeruli were always atypical glomeruli (characterized by their strong degree of cholinergic innervation). In addition to the morphological findings, our biochemical data revealed that olfactory deprivation caused a decrease in the content of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid in the ipsilateral MOB in comparison to the contralateral and control MOBs, together with an increase in noradrenaline levels in both the ipsilateral and contralateral MOBs. Our results show that regulation of the noradrenergic centrifugal system in the MOB depends on environmental olfactory stimulation and that it is highly reactive to sensory deprivation. By contrast, the cholinergic system is fairly stable and does not exhibit clear changes after the loss of sensory inputs.

Septal organ of Grüneberg is part of the olfactory system

The olfactory system in rodents and many other mammals is classically divided into two anatomically separate, and morphologically distinct, sensory systems: the main olfactory system and the accessory olfactory system. We have now identified a novel third population of olfactory marker protein-expressing sensory neurons that is located in a discrete pocket of the rostral nasal septum, which we refer to as the septal organ of Grüneberg (SOG). Neurons in this region of the septum are located in the submucosa, in small grape-like clusters, rather than in a pseudostratified neuroepithelium, as seen in both the olfactory and vomeronasal neuroepithelia. Despite their unusual location, axons projecting from the SOG neurons fasciculate into several discrete bundles and terminate in a subset of main olfactory bulb glomeruli. These glomeruli most likely represent a subset of atypical glomeruli that are spatially restricted to the caudal main olfactory bulb. The unique rostral position of the SOG suggests that the SOG may be functionally specialized for the early detection of biologically relevant odorants.

Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb

The centrifugal systems innervating the olfactory bulb are important elements in the functional regulation of the olfactory pathway. In this study, the selective innervation of specific glomeruli by serotonergic, noradrenergic and cholinergic centrifugal axons was analyzed. Thus, the morphology, distribution and density of positive axons were studied in the glomerular layer of the main olfactory bulb of the rat, using serotonin-, serotonin transporter- and dopamine- β-hydroxylase-immunohistochemistry and acetylcholinesterase histochemistry in serial sections. Serotonin-, serotonin transporter-immunostaining and acetylcholinesterase-staining revealed a higher heterogeneity in the glomerular layer of the main olfactory bulb than previously reported. In this sense, four types of glomeruli could be identified according to their serotonergic innervation. The main distinctive feature of these four types of glomeruli was their serotonergic fibre density, although they also differed in their size, morphology and relative position throughout the rostro-caudal main olfactory bulb. In this sense, some specific regions of the glomerular layer were occupied by glomeruli with a particular morphology and a characteristic serotonergic innervation pattern that was consistent from animal to animal. Regarding the cholinergic system, we offer a new subclassification of glomeruli based on the distribution of cholinergic fibres in the glomerular structure. Finally, the serotonergic and cholinergic innervation patterns were compared in the glomerular layer. Sexual differences concerning the density of serotonergic fibres were observed in the atypical glomeruli (characterized by their strong cholinergic innervation). The present report provides new data on the heterogeneity of the centrifugal innervation of the glomerular layer that constitutes the morphological substrate supporting the existence of differential modulatory levels among the entire glomerular population.

A sexually dimorphic group of atypical glomeruli in the mouse olfactory bulb.

Atypical glomeruli (AtG) are clearly distinguishable from typical ones because of their strong cholinergic innervation. AtG are located in defined positions in the caudal half of the main olfactory bulb of rodents. The AtG partially overlap with other specialized olfactory subsystems, such as the modified glomerular complex, which is close to the accessory olfactory bulb. So far, possible sex differences in these specialised olfactory systems have not been investigated. In this work we have identified AtG in the mouse by means of acetylcholinesterase histochemistry and compared the number and size of these glomeruli between the sexes and also between the two strains that demonstrate intraglomerular synaptic differences, i.e. BALB/c and CD-1 mice. First, we divided the AtG into three types according to their position (I, rostral-most; II, around the accessory olfactory bulb; III, caudal-most) or their reactivity to acetylcholinesterase histochemistry (AtG type II being the least reactive glomeruli). ANOVA analyses revealed differences in the maximum diameter of glomeruli among the three types, but not in their sectional areas, indicating that all three types have different shapes. Moreover, both morphoplanimetric parameters were seen to be different between the two strains studied and also between the sexes: male mice and BALB/c animals had the largest glomeruli. The number of AtG was also significantly different between the sexes and strains, although these factors presented a strong interaction. Thus, the males had higher numbers of AtG in the CD-1 strain whereas in the BALB/c mice males demonstrated fewer AtG than females. These differences in number were largely due to AtG type II. The present work is evidence that AtG type II is a sexually dimorphic group of specialized glomeruli located in the main olfactory bulb.

Bilateral olfactory deprivation reveals a selective noradrenergic regulatory input to the olfactory bulb

Unilateral olfactory deprivation in the rat induces changes in the catecholaminergic system of the olfactory bulb. Nevertheless, evidence suggests that unilateral deprivation does not fully prevent stimulation of the deprived bulb. The present report analyses the response of the catecholaminergic system of the olfactory bulb in fully deprived rats obtained by bilateral naris occlusion. The complete deprivation produces more rapid and dramatic changes in both the intrinsic and extrinsic catecholaminergic systems of the olfactory bulb. Intrinsic responses involve a rapid decrease in dopamine-containing cells to about 25% of controls, correlated with a decreased Fos expression in juxtaglomerular cells of all olfactory glomeruli, with the only exception of those of the atypical glomeruli which maintain unaltered expression of both markers. In parallel with these events, there is a progressive increase in the density of extrinsic noradrenergic axons arising from neurons in the locus coeruleus, which shows, in parallel, a progressive increase in Fos expression. This model demonstrates plastic changes in the catecholaminergic system of the olfactory bulb forming a valid morphological substrate for lowering thresholds in the processing of olfactory information. In addition to this generalized response, there is another one, directed to a specific subset of olfactory glomeruli (atypical glomeruli) involved in the processing of odor pheromone-like cues related to behavioral responses, that could be responsible for keeping active this reduced and selected group of glomeruli carrying crucial olfactory information. These results indicate the existence of adaptive changes in the catecholaminergic system of the olfactory bulb as a response to the lack of afferent peripheral stimulation. These changes involve dopamine- and noradrenaline-immunoreactive elements, in a strategy presumably directed at maintaining to the highest possible level the ability to detect olfactory signals.

Olfactory bulb and retrobulbar regions in the hedgehog tenrec: Organization and interconnections

The Madagascan lesser hedgehog tenrec (Echinops telfairi) is a terrestrial, nocturnal insectivore with a low encephalization index and a huge olfactory bulb. To gain insight into the organization and evolution of olfactory regions in placental mammals, the cytoarchitecture (Nissl), neurochemical attributes [zinc and acetylcholinesterase stain, nicotinamide adenine dinucleotide phosphate (NADPh)-diaphorase, and calcium-binding proteins], and interconnections (injections of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran amine) of tenrec bulbar and retrobulbar regions were examined. The tenrec has a well-laminated main olfactory bulb, and modified (atypical) glomeruli are found that, to date, have been demonstrated only in murine rodents. Compared with the main olfactory bulb, the accessory bulb is relatively small, with clearly different staining characteristics, particularly with respect to NADPh-diaphorase, anticalbindin, and anticalretinin. External and central anterior olfactory nuclei also show characteristic cytoarchitectural and chemoarchitectural features. The medial olfactory peduncle seems to differ considerably from that in rodents. A small taenial structure can be separated from the hippocampal continuation. This taenia tecti presumably corresponds to the superior part of the tenia tecti in rodents, but no homologue of the rodent's prominent inferior taenia tecti could be found. The connections of bulbar and retrobulbar regions are similar to those seen in other mammals. Interbulbar projection systems connect the two olfactory bulbs through an external (topographic) and central (nontopographic) anterior nucleus; however, the topographic arrangement of the intrabulbar association system seems to differ from that seen in rodents. A reciprocity of direct olfactory bulb connections with the frontal (sulcal/orbital) cortex was found in the tenrec that has not been reported so far in other species.

Distribution of acetylcholinesterase and choline acetyltransferase in the main and accessory olfactory bulbs of the hedgehog (Erinaceus europaeus).

The distribution of cholinergic markers was studied in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) of the western European hedgehog (Erinaceus europaeus) by using choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. A dense network of AChE-containing and ChAT-immunoreactive fibers was observed innervating all layers of the MOB except the olfactory nerve layer, where neither AChE- nor ChAT-labeled elements were found. The highest density of AChE- and ChAT-positive axons was found in the glomerular layer (GL)/external plexiform layer (EPL) boundary, and in the internal plexiform layer. This general distribution pattern of ChAT- and AChE-stained axons resembled the distribution pattern found in rodents. Nevertheless, some interspecies differences, such as the lack of atypical glomeruli in the hedgehog, were also found. In addition to fibers, a population of noncholinergic and presumably cholinoceptive AChE-active neurons was observed in the hedgehog. All mitral and tufted cells of the hedgehog MOB showed a dark AChE staining unlike previous observations in the mitral and tufted cells of rodents. As in other species previously reported, subpopulations of external tufted cells and short-axon cells were also AChE-active. Finally, a population of small AChE-containing cells was observed in the EPL of the hedgehog MOB. The size, shape, and location of these cells coincided with those of satellite and perinidal cells, two neuronal types described previously in the EPL of the hedgehog and not present in the rodent MOB. The AOB of the hedgehog showed a distribution of AChE- and ChAT-positive fibers similar to the rodent AOB. Nevertheless, a heterogeneous innervation of vomeronasal glomeruli by bundles of AChE- and ChAT-labeled axons found in the hedgehog has not been previously found in any other species. As in the MOB, all mitral cells in the AOB showed a strong AChE activity. These results demonstrate some similarities but also important differences between the distribution of ChAT and AChE in the MOB and AOB of rodents and this primitive mammalian. These variations may indicate a different organization of the cholinergic modulation of the olfactory information in the insectivores.

Segregated distribution of TH-immunoreactivity in olfactory glomeruli.

Atypical and typical olfactory glomeruli differ in their primary afferents, centrifugal control and in some chemically identified subpopulations of interneurones. The distribution of tyrosine hydroxylase (TH)-immunopositive neurones in the periglomerular region of both typical and atypical glomeruli has been studied using a double histochemical-immunohistochemical method. A segregated distribution of TH-immunopositive cells was found among both types of glomeruli. TH-immunolabelled cells were more abundant (p < 0.05) in the atypical glomeruli. These data suggest that some neuronal subpopulations are related to specific properties of the glomerular physiology and they have a segregated distribution in different subsets of glomeruli. Thus, catecholamines might be involved in the processing of specific olfactory cues in atypical glomeruli. This study presents new differences in the cellular composition of typical and atypical glomeruli.

Calcium-binding proteins in the periglomerular region of typical and atypical olfactory glomeruli

The distribution of chemically identified neuronal populations was studied in the glomerular layer of the rat olfactory bulb using calcium-binding protein immunocytochemistry combined with acetylcholinesterase histochemistry. Four calcium-binding proteins (calbindin D-28k, parvalbumin, calretinin, and neurocalcin) were analyzed in the periglomerular region of two different glomerular subsets: typical and atypical glomeruli. Atypical glomeruli were clearly distinguishable from typical ones by their dense network of acetylcholinesterase-positive centrifugal fibers. Each calcium-binding protein studied showed a specific distribution pattern in the rat olfactory bulb. Calbindin D-28k-, calretinin-, and neurocalcin-immunoreactive neurons were specially abundant in the glomerular layer. These three calcium-binding proteins had their main expression in neuronal subpopulations directly involved in the glomerular circuitries of the rat olfactory bulb. Specific populations of periglomerular cells were stained for calbindin D-28k, parvalbumin, calretinin, or neurocalcin, whereas external tufted cells were only immunoreactive to neurocalcin. Both neuronal types, periglomerular cells and external tufted cells, were found in the periglomerular region of both glomerular subsets. Nevertheless, a homogeneous distribution of calbindin D-28k- or calretinin-immunopositive periglomerular cells were found between typical and atypical glomeruli, whereas the neurocalcin-immunostained external tufted cells were statistically more abundant in typical glomeruli than in atypical ones ( P<0.001). These data suggest that some neuronal subpopulations are related with general properties of the glomerular physiology, and they have a homogeneous distribution in different subsets of glomeruli, whereas other chemically identified populations are related with a finer tuning of the olfactory processing, and they are segregately distributed in relation to particular glomerular subsets. In addition, this work adds new differences in the cellular composition of typical and atypical glomeruli.

Segregated distribution of nitric oxide synthase-positive cells in the periglomerular region of typical and atypical olfactory glomeruli

Using double-staining techniques, the distribution of NADPH-diaphorase (ND)- and nitric oxide synthase (NOS)-positive cells was compared in the periglomerular region of typical and atypical rat olfactory glomeruli. Dorsomedial and ventrolateral atypical glomeruli contained similar number of ND/NOS-positive periglomerular cells. The number of ND/NOS-stained periglomerular cells was much higher ( P < 0.001) in typical than in atypical glomeruli. The present results indicate that, in addition to described neurochemical and ultrastructural differences between the neuropile of typical and atypical glomeruli, there are also marked differences in the phenotype of periglomerular interneurons of both glomerular subsets.

Atypical olfactory glomeruli subset of the rat: quantitative study and organization of the peripheral afferents.

The atypical glomeruli constitute a particular subset of olfactory glomeruli in the rat olfactory bulb which is mainly characterized by a strong centrifugal cholinergic innervation. In the present study, the topographical organization of the mucoso-bulbar projection of these glomeruli was analysed using small injections of WGA-HRP into the anterior nasal cavity of adult rats. The atypical olfactory glomeruli were visualized on adjacent bulbar sections using acetylcholinesterase histochemistry. A mean of 29 atypical glomeruli per bulb was observed in several areas of the posterior half of the olfactory bulb. Following the rostro-caudal axis of the olfactory bulb, the first atypical glomeruli were located in lateral positions, then in dorsal and ventral ones. The most posterior atypical glomeruli were located in the bulbar medial side. Concerning the projections from the periphery to the atypical glomeruli, various WGA-HRP patterns of labelling were observed. When the surface area of injection sites in the anterior part of the olfactory sheet was between 30 and 40 mm2, half of the atypical population was labelled with the atypical glomeruli being heavily labelled. All sites of distribution previously described were represented. When the surface area of injection sites was inferior to 20 mm2, only some positions distributed along the bulbar antero-posterior axis were represented. These atypical glomeruli were generally partially labelled. Taken together, these results suggest that, although atypical glomeruli are restricted in the posterior olfactory bulb, they receive peripheral projections diffusely organized along the antero-posterior axis of the olfactory mucosa. This profile was compared with that of other classical olfactory glomeruli.

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (α4β2-like) and bungarotoxin-sensitive (α7-like) receptors were visualized using [ 3H]pirenzepine, [3H]AF-DX 384, [ 3H]cytisine and [ 125I]α-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [ 3H]vesamicol (vesicular acetylcholine transport sites) and [ 3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [ 3H]vesamicol and [ 3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [ 125I]α-BTX binding sites were mostly concentrated in the superficial bulbar layers, [ 3H]cytisine binding was found in the glomerular layer, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [ 3H]vesamicol and [ 3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [ 3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/α4β2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.

Postnatal development of cholinergic markers in the rat olfactory bulb: A histochemical and immunocytochemical study

The present study has defined the developmental time course and the distribution patterns of neuronal fibers and cell bodies displaying acetylcholinesterase (AChE) activity or choline acetyltransferase (ChAT) immunoreactivity in the rat olfactory bulb. The results indicate that the deployment of centrifugal AChE-containing fibers is essentially postnatal. The subset of atypical glomeruli, including the modified glomerular complex, is innervated as early as the first postnatal day while the normal ones are not reached by this type of afferent before postnatal day 6. The comparison of AChE labelling with ChAT immunoreactivity strongly supports the assumption that AChE-containing fibers represent mainly, if not exclusively, the cholinergic bulbopetal innervation emanating from the basal forebrain. A quantitative study has confirmed that the density of labelled fibers increases gradually in the postnatal period and spreads heterogeneously among the bulbar layers. The selective precocious innervation of atypical glomeruli is in favor of their involvement in the early processing of olfactory information in the olfactory bulb. Acetylcholinesterase is also expressed within a subset of ChAT-negative interneurons of the developing olfactory bulb. The number of neurons expressing AChE increases from birth to postnatal day 15 and then decreases to reach the adult value on about postnatal day 30. This neuronal population could constitute a cholinoceptive subset mediating the effects of cholinergic afferents on the bulbar neuronal network.

Comparison of necklace olfactory glomeruli with atypical glomeruli in the rat olfactory bulb

Comparison of necklace olfactory glomeruli with atypical glomeruli in the rat olfactory bulb

Atypical olfactory glomeruli contain original olfactory axon terminals: An ultrastructural horseradish peroxidase study in the rat

The labelling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity has been studied in the rat. In such conditions, atypical glomeruli, previously described according to their high acetylcholinesterase content, display a strong tracer accumulation. The course of afferent olfactory fibres could be followed along the lateral and dorsal surface of the olfactory bulbs. The primary olfactory axons ending in atypical glomeruli have been identified with horseradish peroxidase in electron microscopy. They differ significantly from classical olfactory terminals owing to the presence of large dense-cored vesicles accompanying small clear ones. Moreover, the olfactory terminals do not gather in dark nodules as they do classically in olfactory glomeruli. The study demonstrates that a subset of olfactory neuroreceptors displaying original ultrastructural characteristics projects selectively into atypical olfactory glomeruli. Ultrastructural features indicate that olfactory information processing taking place in the neuropil might be similar to that which occurs in typical glomeruli. Considered together, the atypical olfactory neuroreceptors, glomeruli and acetylcholinesterase-containing centrifugal fibres could constitute a new olfactory subsystem. This hypothesis is discussed by taking into account previous demonstration of other olfactory subsystems devoted to the processing of olfactory cues of fundamental biological importance.