The morphology and topographic distribution of substance-P-like immunoreactive amacrine cells in the cat retina

In cat retinal wholemounts, substance-P-like immunoreactivity (SP-IR) was localized in a distinct population of amacrines whose cell bodies were normally placed in the ganglion cell layer. Although displaced amacrines accounted for 80-95% of the SP-IR amacrines in peripheral retina, this proportion decreased considerably within the area centralis, accounting for 50-80% of the labelled cells at maximum density. The SP-IR cells in both the inner nuclear and ganglion cell layers gave rise to well-defined varicose dendrites of uniform appearance that stratified around 60% depth (S3/S4) of the inner plexiform layer. In addition, sparse fine dendrites in stratum 1 (S1) could sometimes be traced to inner nuclear cells and occasionally to displaced amacrines. The combined SP-IR cell density ranged from less than 50 cells mm-2 in the far periphery to more than 500 cells mm-2 in the area centralis; the maximum density showed little individual variation despite wide differences in the proportion of displaced cells. The 39,000 SP-IR amacrines in a mapped retina had a triangular topographic distribution, with intermediate isodensity lines extending vertically in superior retina and horizontally along both arms of the visual streak. Colocalization experiments established that all SP-IR cells in cat retina showed GABA-like immunoreactivity, and that the SP-IR amacrines were quite distinct from the cholinergic amacrines identified by choline acetyltransferase immunohistochemistry.     

Prolonged expression of puma in cholinergic amacrine cells during the development of rat retina

During development of the nervous system, large numbers of neurons are overproduced and then eliminated by programmed cell death. Puma is a BH3-only protein that is reported to be involved in the initiation of developmental programmed cell death in rodent retinal neurons. The expression and cellular localization of Puma in retinal tissues during development are not, however, well known. Here the authors report the expression pattern of Puma during retinal development in the rat. During the period of programmed cell death in the retina, Puma was expressed in some members of each retinal neuron, including retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptor cells. Although the developmental programmed cell death of cholinergic amacrine cells is known to be independent of Puma, this protein was expressed in almost all their dendrites and somata of cholinergic amacrine cells at postnatal age 2 to 3 weeks, and it continued to be detected in cholinergic dendrites in the inner plexiform layer for up to 8 weeks after birth. These results suggest that Puma has some significant roles in retinal neurons after eye opening, especially that of cholinergic amacrine cells, in addition to programmed cell death of retinal neurons before eye opening.     

Appearance of immunoreactive endocrine cells during the development of the rat pancreas, with special reference to polypeptide-secreting cells

The chronological appearance of PP cells in fetal pancreatic islets was studied using specific anti-PP serum and the direct peroxidase method. The presence of A and B cells was also studied, using the same immunocytochemical technique, as a reference pattern related to data previously reported. Our data confirm that the A cell is the earliest endocrine cell type, appearing on the 12th day of gestation, followed by B cells (14th day) and later on by PP cells (19th day). Primitive islets were identified in the pancreas after the 15th day. However, the spatial cell disposition observed in the adult islet was only recognized at the 20th day of gestation. The data reported provide the necessary information to establish the complete chronology in the rat fetus. Consequently, the development of pancreatic islets in the rat fetus could be employed as a useful model to study the existence of factors that control the sequential appearance of endocrine cells and the possible changes occurring in the islets of animals with genetic diabetes during the fetal period.     

Structure of the non-lymphoid cells during the postnatal development of the rat lymph nodes

This study describes the postnatal development of the nonlymphoid cells with special reference to the fibroblastic reticulum cells (FRCs) and interdigitating cells (IDCs). The first lymphocytes of the neonatal lymph nodes are located in the developing deep cortex units (DCUs) identified by the Gomori's technique for reticulin fibres. Ultrastructural studies demonstrate that FRCs form the stroma of the DCUs. By light and electron microscopy, it is demonstrated that FRCs occupy the outer cortex in the following stages of development of the lymph nodes. Thus, FRCs form the stroma of the primary follicles and, later, are transformed in follicular dendritic cells (FDCs) of the germinal centres. Immature or pro-IDCs appear as migrating elements in the deep cortex of lymph nodes of the neonatal rats. The ultrastructure of the pro-IDCs resembles that of the mature IDCs but not that of the phagocytic cells. Pro-IDCs are transformed into mature IDCs whose cytoplasmic expansions contact lymphocytes via tight junctions. Some of these lymphocytes are likely apposed to FRCs of the DCUs. No cells containing Birbeck granules were found in the parenchyma of the lymph nodes during the postnatal development. The role of these nonlymphoid cells is discussed with respect to the immunologic function of mammalian lymph nodes.     

Modulation of synaptic function in retinal amacrine cells

Amacrine cells are interneurons that have diverse functionsin retinal signal processing. In order to study signaling andmodulation in retinal amacrine cells, we employ a simplifiedculture system containing identifiable GABAergic amacrine cells.Immunocytochemistry experiments indicate that GABAergic amacrinecells express metabotropic glutamate receptor 5 (mGluR5), agroup I mGluR usually linked to the IP3 signaling pathway. Ca²⁺imaging experiments using an mGluR5-specific agonist indicatethat these receptors are functional and when activated, canstimulate temporally diverse Ca²⁺ elevations. To begin to establishthe role of these receptors in modulating amacrine cell function,we have used electrophysiological methods to ask whether ionchannels are the targets of mGluR5-dependent modulation. Herewe discuss our results indicating that activation of mGluR5leads to enhancement of currents through GABA_A receptors. Thisenhancement is dependent upon elevations in cytosolic Ca²⁺ andactivation of protein kinase C (PKC). To explore the consequencesof Ca²⁺ elevations in another context, we have used nitric oxide(NO) donors to mimic the effects of activating the Ca²⁺-dependentsynthetic enzyme for NO, neuronal nitric oxide synthase. Wefind that exposure to NO donors also enhances the amplitudeof currents through GABA_A receptors. Together, these resultsindicate that glutamate from presynaptic bipolar cells has thepotential to work through multiple mechanisms to regulate thefunction of amacrine-to-amacrine cell GABAergic synapses.     

Polysomes during early postnatal development of brain in the rat

We have attempted to show eventual modifications in the brain protein synthesis apparatus of rat during the first three weeks after birth. Through this time we noted a steady decrease (about 60%) in the free polysomes, when expressed relative to tissue weight. This decrease does not correlate with changes in the polysome profile, indicating that no loss in the efficiency of protein synthesis was involved. Translation in a reticulocyte lysate also failed to reveal differences.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat

Summary A modification of Weiser's (1973) cell isolation method was used in order to study the developmental pattern of various intestinal enzyme activities in villus and crypt cells of normal rats from 5 days after birth until 8 weeks. Alkaline phosphatase and enterokinase activities were always located in the upper villus zone during postnatal development. Enterokinase activity was higher in the upper villus cells during the third week of life than after this period. Aminopeptidase activity was located in the crypt cells during the first week, its maximum activity remained in this area until the third week. At this time, sucrase activity appeared in the crypt cells, then aminopeptidase and sucrase activities rose to the villus zone during the fourth week. Amylase activity was detected along the entire crypt-villus axis 5 days after birth, reaching maximum activity in crypt cells at the end of the first week and in the upper villus cells after the fourth week. In contrast with the other enzymes studied almost all amylase activity was soluble in the youngest animals whereas at weaning most of the activity appeared in a particulate form in the villus cells. But in the crypt cells the ratio between particulate and soluble form remained unchanged until the adult stage. Various hypotheses are advanced to explain the patterns of evolution of the different enzymes.     

Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

Browning attenuates murine white adipose tissue expansion during postnatal development

During postnatal development of mice distinct white adipose tissue depots display a transient appearance of brown-like adipocytes. These brite (brown in white) adipocytes share characteristics with classical brown adipocytes including a multilocular appearance and the expression of the thermogenic protein uncoupling protein 1. In this study, we compared two inbred mouse strains 129S6sv/ev and C57BL6/N known for their different propensity to diet-induced obesity. We observed transient browning in retroperitoneal and inguinal adipose tissue depots of these two strains. From postnatal day 10 to 20 the increase in the abundance of multilocular adipocytes and uncoupling protein 1 expression was higher in 129S6sv/ev than in C57BL6/N pups. The parallel increase in the mass of the two fat depots was attenuated during this browning period. Conversely, epididymal white and interscapular brown adipose tissue displayed a steady increase in mass during the first 30 days of life. In this period, 129S6sv/ev mice developed a significantly higher total body fat mass than C57BL6/N. Thus, while on a local depot level a high number of brite cells is associated with the attenuation of adipose tissue expansion the strain comparison reveals no support for a systemic impact on energy balance. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.     

Properties of mouse retinal ganglion cell dendritic growth during postnatal development

The property of dendritic growth dynamics during development is a subject of intense interest. Here, we investigated the dendritic motility of retinal ganglion cells (RGCs) during different developmental stages, using ex vivo mouse retina explant culture, Semliki Forest Virus transfection and time-lapse observations. The results illustrated that during development, the dendritic motility underwent a change from rapid growth to a relatively stable state, i.e., at P0 (day of birth), RGC dendrites were in a highly active state, whereas at postnatal 13 (P13) they were more stable, and at P3 and P8, the RGCs were in an intermediate state. At any given developmental stage, RGCs of different types displayed the same dendritic growth rate and extent. Since the mouse is the most popular mammalian model for genetic manipulation, this study provided a methodological foundation for further exploring the regulatory mechanisms of dendritic development.     

Trophic responsiveness of purified postnatal and adult rat retinal ganglion cells

Central neurons lose the ability for axonal re-growth during development and typically do not regenerate their axons following axotomy once they become mature unless given a growth-permissive environment i.e. peripheral nerve graft. In the present study, the growth responsiveness of purified retinal ganglion cells (RGCs) at different ages to neurotrophic factors and Schwann cell (SC)-secreted factors were examined directly. The purity of adult RGCs was 97% as assessed by retrograde labelling with 4,6-diamidino-2-phenylindole. The stability of cultures were demonstrated by long-term survival (30 days) in medium contained brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin (F) (BCF). RGCs from postnatal (P) (P0, P4, P8, P21) and adult (P90) rats showed decreasing levels of survival and neuritogenesis when grown in BCF. In contrast, the opposite was observed in SC-conditioned medium (CM)-treated P0-P8 RGCs which were increasingly responsive. SCCM induced maximal neurite outgrowth in P8 RGCs via the activation of extracellular regulated kinase 1/2 (Erk1/2). Inhibition of mitogen-activated protein kinase-Erk1/2 signaling using an Erk1/2-specific inhibitor (UO126) abolished SCCM-induced Erk1/2 phosphorylation and neuritogenesis completely. Although both SCCM and BCF failed to sustain the same levels of growth in P21 or P90 cultures as observed in P8 cultures, SCCM promoted higher survival and neuritogenesis than BCF-treated adult RGCs. This study is the first report of adult rat RGC purification and demonstrates that mature RGCs need multiple factors for survival and neurite outgrowth.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.     

Differential distribution of a second type of tyrosine hydroxylase immunoreactive amacrine cell in the chick retina

A second population of tyrosine hydroxylase-immunoreactive amacrine cells was demonstrated in embryonic and adult chicken retinas by immunohistochemistry techniques in whole flat-mount preparations. The populations were differentiated on a basis of different immunostaining intensities, levels of stratification in the inner plexiform layer, and topographical distributions. Cells of one type were similar to the previously described dopaminergic amacrine cells, denoted here as tyrosine hydroxylase type 1 cells. Immunoreactive neurons of the second type observed in the present work had relatively smaller somata size, and weaker immunostaining than type 1 cells, and were located preferentially in the ventral retina. These tyrosine hydroxylase type 2 cells could be visualized from embryonic day 14 to 21 days after hatching animals. The distribution of the second population was coincident with that of the targets of centrifugal fibres and with cells involved in long proprioretinal connections. We propose that the tyrosine hydroxylase type 2 amacrine cells found in the ventral retina could mediate an important pathway to the upper half of the visual field so as to aid in the detection of predators.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Summary Changes in -amylase (-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of -amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.Career scientist of the Consejo Nacional de Investigaciones Científicas y Téchnicas of Argentina