Postnatal development of calcium-binding proteins calbindin and parvalbumin in human visual cortex

In adult primate visual cortex, the calcium-binding proteins calbindin (CB) and parvalbumin (PV) are localized in different subsets of GABAergic neurons with a characteristic laminar distribution. However, the emergence and development of CB and PV in relation to the periods of functional maturation of the human visual cortex are not known. Therefore, we examined (i) postnatal changes in the distribution of immunoreactivity (ir) for CB and PV in the visual cortex; (ii) the pattern of changes in immunoreactivity in relation to the synaptic maturation; and (iii) differences in the maturation of CB and PV immunoreactivity between areas 17 and 18. We found a consistently high expression of CB in neonatal visual cortex, particularly in layer IV and infragranular layers. However, despite an early appearance of PV, its peak in development occurred only after 2 months of age, characterized by a transient overexpression in the thalamo-recipient layer IV and a continuous inside-out maturation in supragranular layers. The neonatal pattern of high CB-ir in layers IV-VI was transformed during infancy and childhood into an adult pattern of high CB-ir in layer II, but low CB-ir in layer IV and infragranular layers. There was no difference in pattern and tempo of maturation of calcium-binding proteins between area 17 and 18, indicating simultaneous development of cortical inhibitory circuits among cytoarchitectonically and functionally distinct cortical areas. In addition, the reorganization of CB/PV expression temporally and spatially coincides with the course of cortical synaptogenesis, and delineates the major stages of maturation of the human visual cortex.     

Differences between guinea pig and rat in the dorsal cochlear nucleus: expression of calcium-binding proteins by cartwheel and Purkinje-like cells

This study describes differences between guinea pig and rat in the immunoreactivities for calbindin (CB-IR) and parvalbumin (PV-IR) in cartwheel (CWC) and Purkinje-like (PLC) cells of the dorsal cochlear nucleus (DCN). CWCs are the most important inhibitory interneurons of the DCN. Their soma and dendrites stain intensely for CB-IR in guinea pigs but only weakly and incompletely in rats. In both species, the CWCs do not show PV-IR. PLCs, a rare type of DCN cells often interpreted as displaced cerebellar Purkinje cells misrouted during migration, are known from rat and mouse and are here described for guinea pig DCN. PLCs are intensely and completely stained for CB-IR and PV-IR in guinea pigs. In rats, they stain with similar completeness only for CB-IR, PV-IR being weak and restricted to the cell's soma. Similar staining differences between the two species are seen with the cerebellar Purkinje cells, i.e., PLCs resemble the cerebellar Purkinje cells more than do the CWCs. Based on the present material (and preliminary findings in a primate (marmoset), we speculate that the PLCs have their place in the circuitry of the DCN receiving input via parallel fibers, like the CWCs, and possibly projecting their axon onto the cerebellum.     

Neurotoxic effect of cisplatin and the cisplatin-procaine complex DPR studied in organotypic cultures of chick embryonic dorsal root ganglia

Neurotoxic effects of cisplatin and the cisplatin-procaine complex cis-diaminechloro-[2-(diethylamino)ethyl 4-aminobenzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate (DPR) were compared in organotypic cultures of chick embryonic dorsal root ganglia maintained in a semi-solid (soft agar) culture medium. The changes of two characteristics of the neurite outgrowth, the mean radial length of neuritic processes growing out from the ganglia and the area of neurite outgrowth around the ganglion, were used as parameters to evaluate the toxic effect of both compounds. The drugs were administered to the cultures at concentrations ranging from 13 to 120 microM. The half-maximum inhibition concentration (IC50) was determined from the concentration-response curves for both the mean radial length of neurites and the area of neurite outgrowth. An analysis of these parameters revealed that DPR was significantly less neurotoxic than cisplatin. In fact, considering the mean radial length of neurite processes, the IC50s of cisplatin were 56, 65 and 66 microM after 24, 48 and 72 h of exposure, respectively. By contrast, for DPR the IC50s were 116 microM after 24 h, and greater than 120 microM after 48 and 72 h of exposure. When we considered the area index (i.e. the area of neurite outgrowth normalized for the area of the ganglia), the IC50s for cisplatin were 41, 52 and 55 microM after 24, 48 and 72 h of exposure, respectively, whereas for DPR the IC50s were 59 microM after 24 h, and greater than 120 microM after 48 and 72 h of exposure. Our results support previous findings of lower toxicity of DPR to non-neoplastic tissues, as compared to cisplatin.     

Myelination in organotypic cultures of sympathetic ganglia

Explants of mouse superior cervical ganglion (SCG), co-cultured with dorsal spinal cord, were grown for up to 4 weeks in vitro. In such cultures, scattered internodes of peripheral nervous system (PNS) myelin were observed, apparently associated with SCG neurites. Although rare, the incidence of PNS myelination in this system might merit further experimentation to provide a model facilitating the evaluation of postganglionic sympathetic myelination, which in vivo may be both extensive and morphologically unusual.     

Neurons in the rat occipital cortex co-expressing the substance P-receptor and GABA: a comparison between in vivo and organotypic cultures

The morphology and the distribution of neurons expressing the NK1-receptor (NK1R) and the co-expression of gamma-aminobutyric acid (GABA) in these neurons were studied in the rat occipital cortex and in organotypic cultures (OTCs) derived from this structure. By employing immunohistochemistry, we demonstrate that the NK1R-expressing neurons are non-pyramidal neurons and co-express GABA. Some differences were noted between in vivo and OTCs. NK1R-expressing neurons in OTCs had larger somata and longer dendrites and the proportion stained with an anti-GABA-antibody (approximately 50%) was smaller than in vivo (90%). The preferential location of NK1R-expressing neurons in layers II/III and VI, seen in vivo is not present in OTCs where these neurons distribute rather homogeneously through layers II-VI. Our findings imply that in contrast to the cat and monkey, in the rat occipital cortex the effects of substance P are almost exclusively mediated via inhibitory interneurons.     

Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18-39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP-containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA-immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker.     

Rhythm generation in organotypic medullary cultures of newborn rats

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO2 and H+. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 microM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 microM) and/or hydroxysaclofen (200 microM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 microM) abolished rhythmicity. (3) While atropine sulphate (5 microM) was ineffective in modulating periodic discharges nicotine (100 microM) - like CO2-shortened the intervals between the periodic events; hexamethonium (50-100 microM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 microM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-50 microM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50-100 microM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO2/acidosis. (6) Dantrolene (10 microM). an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 microM) which blocks endoplasmic Ca(2+)-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)-(7) indicate that transmembrane calcium fluxes as well as intracellular Ca(2+)-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.     

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5-10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in approximately 2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.     

Morphological organization of the globus pallidus-subthalamic nucleus system studied in organotypic cultures

The morphological organization of the globus pallidus (GP), the subthalamic nucleus (STN), and the pallidosubthalamic projection was studied in organotypic cultures. Coronal slices from the GP, the STN, the striatum (CPu), and the cortex (Cx) were taken from the rat after postnatal days 0-2 and grown for 2 or 5-6 weeks. For analysis, immunocytochemistry against glutamate (GLU), parvalbumin (PV), and calretinin (CR) was combined with confocal microscopy. After 2 weeks in vitro, the STN showed a densely packed, homogeneous GLU-immunoreactive (ir) cell population. Pallidal GLU-ir neurons were heterogeneous, consisting of large-sized weakly GLU-ir neurons and small-sized intensively GLU-ir neurons. After 5-6 weeks in vitro, pallidal axons had radiated from numerous large-sized PV-ir cells and selectively innervated the STN, where they heavily ramified. Cultured STN neurons were not stained for PV; however, multipolar intensely PV-ir neurons were located at the border of the STN with their dendrites oriented towards the STN. Double labeling for PV and CR in both mature cultures and in the adult rat revealed that the culture CR-ir neurons from the GP, the Cpu, and from areas adjacent to the STN were different from cultured PV-ir neurons and their morphologies and distribution corresponded to that in vivo. These results demonstrate that 1) cultured CP and STN neurons display similar morphologies found in in vivo, 2) PV-ir pallidal neurons heavily and selectively innervate the STN; 3) there is a specific class of STN border neurons; and 4) in contrast to the in vivo situation, most cultured STN neurons are PV-negative.     

Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.     

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.     

Development of glycinergic transmission in organotypic cultures from auditory brain stem

We investigated whether glycinergic transmission develops organotypically in auditory brain stem cultures. Slices of the medial nucleus of the trapezoid body and the lateral superior olive were incubated in medium with a raised extracellular K+ concentration. As in vivo, glycine receptor alpha1 subunit immunoreactivity increased and became clustered on somata and proximal dendrites. Together with organotypic expression of glycine transporter GLYT2, this indicates that molecular components of glycinergic synapses form properly. In contrast, glycinergic synaptic currents did not develop as in vivo: after 7 days in vitro they were still similar to those at the time of culture preparation. We suggest that for organotypic development of glycine receptors and transporters, Ca2+ influx due to elevated K+ is sufficient. The development of functional synaptic transmission, however, may require patterned electrical activity.     

Morphological features of neurons containing calcium-binding proteins in the human striatum

An immunohistochemical approach was used to characterize the morphological phenotype of neurons containing the calcium-binding proteins calretinin (CR), parvalbumin (PV), or calbindin-D28k (CB) in the normal human striatum. The protein CR occurs in at least four morphologically distinct types of neurons. Apart from the numerous medium-sized aspiny interneurons and the less abundant giant aspiny interneurons, CR also labels some medium-sized spiny neurons morphologically identical to striatal projection neurons. This finding indicates that CR is not only confined to striatal interneurons but also may be involved in the function of certain projection neurons. Some small and peculiar bushy-like aspiny neurons also are enriched with CR. These neurons could correspond to the dwarf or neurogliform neurons first described by Ramón y Cajal (1911). Three types of PV-immunoreactive striatal neurons can be visualized in the human striatum: 1) the common medium-sized aspiny leptodendritic neurons, 2) some smaller and profusely arborized aspiny neurons, and 3) a few large and intensely stained neurons with conspicuously beaded and poorly branched dendrites. The protein CB labels virtually all medium-sized spiny projection neurons located in the striatal matrix but also identifies a small subset of large and more intensely immunostained aspiny neurons. The latter finding indicates that CB is not entirely confined to striatal projection neurons but also may play a role in local circuit neurons. These normative data should help our understanding of the chemical anatomy of the human striatum in both health and disease.     

Coexistence of calcium-binding proteins in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of the calcium-binding proteins (CaBPs), calbindin D-28k, parvalbumin and S100 protein, were immunohistochemically examined in the glossopharyngeal and vagal sensory ganglia, the carotid body and taste buds. The CaBPs were found in each ganglion with the nodose ganglion containing the largest number of CaBP-immunoreactive (ir) cells (calbindin D-28k > or = S100 > parvalbumin). The coexistence of CaBPs was found in neurons of the nodose, petrosal, and jugular ganglia. Calbindin D-28k-ir neurons in the nodose and petrosal ganglia frequently colocalized S100-ir whereas calbindin D-28k-ir neurons in the jugular ganglion less frequently contained S100-ir. Only small percentages of calbindin D-28k-ir neurons in each ganglion colocalized parvalbumin. Similarly, S100-ir neurons in the nodose and petrosal ganglia frequently colocalized calbindin D-28k-ir whereas S100-ir neurons in the jugular ganglion less frequently contained calbindin D-28k-ir. Moderate to small percentages of S100-ir neurons in each ganglion colocalized parvalbumin. Parvalbumin-ir neurons nearly always colocalized S100-ir in the nodose, petrosal and jugular ganglia. Moderate to small percentages of parvalbumin-ir neurons in each ganglion colocalized calbindin D-28k. Whereas calbindin D-28k- and S100-ir were colocalized in nerve fibers and cells within taste buds of circumvallate papilla of the tongue, the coexistence of these CaBPs could not be determined in the carotid body. These findings suggest a co-operative role for CaBPs in the functions of subpopulations of nodose and petrosal ganglia neurons.     

Relationship of thalamic basal forebrain projection neurons to the peptidergic innervation of the midline thalamus

To better understand the input-output organization of the midline thalamus, we compared the distribution of its peptidergic and monoaminergic afferents, which were visualized by using immunocytochemistry, with the distribution of neurons projecting to different basal forebrain structures, which were mapped using retrograde fluorescent tracers. Serotonin and most of the peptides were found throughout paraventricular thalamic nucleus (PV) and in other midline and intralaminar nuclei (type 1 pattern). Neuropeptide Y, alpha MSH and the catecholamine synthetic enzymes were largely restricted to dorsolateral PV (type 2 pattern). Vasopressin was found in dorsomedial PV and intermediodorsal nucleus in a pattern complementary to the type 2 distribution (type 3 pattern). Neurons projecting to accumbens core were present in paraventricular, intermediodorsal, and other midline nuclei. Neurons projecting to accumbens shell and to central amygdaloid nucleus were found in dorsal PV. The peptidergic zones were only loosely correlated with the distribution of different classes of projection neurons. The type 2 pattern overlapped best with neurons projecting to accumbens shell, and to a lesser extent to central amygdaloid nucleus, while the type 3 pattern overlapped best with neurons projecting to core of accumbens. This partial overlap suggests that some brainstem and hypothalamic nuclei preferentially affect different basal forebrain targets through the midline thalamus, and may allow, for example, information about stress to specifically influence accumbens shell and central amygdaloid nucleus. Nevertheless, most of the peptidergic afferents (type 1 pattern) to midline thalamus cover neurons projecting throughout the basal forebrain, which suggests that all of these neurons receive a variety of brainstem and hypothalamic inputs.     

Expression of the proenkephalin A gene in organotypic cultures of neocortex from newborn rats

It is usually said that axons and nerve terminals do not contain messenger RNA (mRNA) and that peptide transmitters are packaged in granules and transported towards the periphery of the neuron. However, several recent reports challenge this view by showing evidence of the existence of mRNA in axons. In the present study, we demonstrate the existence of mRNA coding for gamma-preprotachykinin-A in rabbit iris by Northern blot analysis and Southern blot analysis of the polymerase chain reaction (PCR)-amplified products. Interestingly, mRNA coding for gamma-preprotachykinin-A was detected also in aqueous humor from eyes exposed to injury (infrared irradiation of the iris or retrobulbar injection of the C-fibre excitant capsaicin), but not from contralateral eyes and normal eyes of untreated rabbits. Our results suggest that the mRNA coding for gamma-preprotachykinin-A occurs in C-fibres in the iris and that it is released into the aqueous humor together with tachykinins in response to C-fibre stimulation.     

Calretinin immunoreactivity in the developing thalamus of the rat: a marker of early generated thalamic cells

The present work was aimed to study the immunocytochemical localization of the calcium-binding protein, calretinin, in the rat thalamus from embryonic day 14 to the third postnatal week. In the adult rat thalamus, calretinin immunoreactivity is intensely expressed in some intralaminar and midline nuclei, as well as in selected regions of the reticular nucleus. At embryonic day 14, calretinin was expressed by immature and migrating neurons and fibres laterally to the neuroepithelium of the diencephalic vesicle in the region identified as reticular neuroepithelium. At embryonic day 16, immunoreactive neurons were present in the primordium of the reticular nucleus and in the region of the reticular thalamic migration, where neurons showed the morphology of migratory cells. At the end of embryonic development and in the first postnatal week, calretinin-positive neurons were observed in selected region of the reticular nucleus and it was intensely expressed in some intralaminar and midline nuclei. Bands of immunopositive fibres were also observed crossing the thalamus. During the second postnatal week, the immunolabelling in the reuniens, rhomboid, paraventricular and central medial thalamic nuclei remains very intense while a decrease of immunoreactivity in mediodorsal, centrolateral and laterodorsal nuclei was observed. The immunostaining of fibres, particularly evident in the perinatal period, progressively decreased and it was no longer visible by the end of the second postnatal week when the distribution and intensity of calretinin immunostaining was similar to that observed in the adult rat thalamus. The present findings indicate that the immunolocalization of calretinin can be used to identify subsets of thalamic neuronal population during pre- and postnatal maturation allowing also the detection of the migratory pattern of early generated reticular thalamic neurons.     

Postnatal development and the differential expression of presynaptic terminal-associated proteins in the developing retina of the Brazilian opossum, Monodelphis domestica

In the present study we have characterized the postnatal (PN) development of the retina in the Brazilian opossum, Monodelphis domestica. Monodelphis, a small, pouchless marsupial, undergoes a protracted period of postnatal development. Using bromodeoxyuridine immunohistochemistry, we have investigated postnatal neurogenesis of the retina. In addition, we have examined the differentiation of the retina by using antibodies directed against the presynaptic terminal-associated proteins synaptotagmin, Rab3A, synaptophysin and synaptosomal-associated protein-25 (SNAP-25), and have characterized their spatial and temporal distribution during postnatal development. This study is the first systematic comparison of the developmental expression of multiple presynaptic terminal-associated proteins in relation to retinal neurogenesis and differentiation. At birth (1PN), the Monodelphis retina was relatively undifferentiated morphologically and birthdating analysis revealed mitotically active cells throughout the retina. The 8PN retina was organized into two cellular layers: an outer region of mitotically active neuroepithelial cells and an inner region of postmitotic cells. The inner plexiform layer formed between 5PN and 10PN, and exhibited unique patterns of immunoreactivity with the antibodies used in this analysis. By 25PN the retina was well laminated, and synaptotagmin-, Rab3A-, synaptophysin- and SNAP-25-like immunoreactivities exhibited distinct and specific patterns within the plexiform layers, although they had not yet achieved their mature, adult patterns. These results indicate that each of these proteins exhibits developmentally regulated changes in its cellular localization, and therefore may play important roles during morphogenesis and synaptogenesis of the vertebrate retina.     

Immunohistochemical analysis of proliferation and differentiation in organotypic cultures of cervical tumor cell lines

Researchers have previously demonstrated that organotypic cultures of cervical tumor cell lines exhibit morphological characteristics similar to the in vivo biopsies from which they were derived (Rader et al., 1990). Both the in vivo biopsy and organotypic culture appeared undifferentiated. We have extended these studies with immunohistochemical analysis using the proliferation and differentiation markers, proliferating cell nuclear antigen (PCNA) and involucrin, respectively, to evaluate in more detail the ability of cervical tumor cell lines to differentiate in organotypic culture. An HPV-immortalized keratinocyte cell line, PE-4, expressed PCNA in the lower half and involucrin in the upper half of the organotypic culture which is consistent with the characteristics of a preneoplastic lesion in vivo. The CC-1 cell line, derived from an invasive squamous cell carcinoma, appeared undifferentiated, but expressed involucrin in the upper half of the organotypic culture. This is the first observation of expression of a differentiation marker in an organotypic culture of a cervical tumor cell line. The other cervical tumor cell lines, SiHa and HeLa, derived from a squamous cell carcinoma, and an adenocarcinoma of the cervix, respectively, did not express detectable levels of involucrin or mucin. All three cervical tumor cell lines, CC-1, SiHa and HeLa, expressed PCNA throughout their entire thickness. The majority of nuclei in SiHa and HeLa cultures were PCNA-positive, while the CC-1 cell line exhibited a lower growth fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatovisceral projections from spinal cord and dorsal column nuclei to the thalamus in hedgehog tenrecs

In order first to overcome the difficulties in understanding the increasing amount of information available regarding the mammalian somatosensory thalamus, and then to correlate the findings among different species and integrate them into a general concept of thalamic organization, the present study investigated the spinothalamic and medial lemniscal projections in Madagascan hedgehog tenrecs (Echinops telfairi and Setifer setosus). Tracer substances were injected into the dorsal column nuclei and into spinal segments at various levels; additional injections were made into the inferior colliculus. The ascending somesthetic projections were to predominantly contralateral posterolateral target areas, and were almost mirror-like on both sides to intralaminar and medial thalamic nuclei. The densest and most extensive projections, originating mainly from the high cervical spinal cord and the dorsal column nuclei, reached the posterolateral thalamus caudal to the lateral geniculate nucleus. This region was difficult to subdivide cytoarchitecturally; nevertheless, on the basis of its labeling pattern, several subdivisions could be described and preliminary named. Some of them compared tentatively with the internal portion of the medial geniculate nucleus (GM) and the ventral posterior nuclear complex (VPC) in more differentiated mammals. The most prominent subdivision, however, located subjacent to the lateral surface of the brainstem, was shown to receive additional fibers from the inferior colliculus. This region might be considered a further subdivision of GM, VPC, a perigeniculate area, and/or a region of its own not comparable at present, with thalamic regions in other mammals. On the other hand, it may also be a remnant of the hypothetical, diffuse multimodal region from which GM and VPC have possibly evolved.(ABSTRACT TRUNCATED AT 250 WORDS)