Development of the delay lines in the nucleus laminaris of the chicken embryo revealed by optical imaging.

One strategy in localizing a sound source in the azimuthal plane is the comparison of arrival times of sound stimuli at the two ears. The processing of interaural time differences (ITDs) in the auditory brainstem was suggested by the Jeffress model in 1948. In chicks, binaural neurons in the nucleus laminaris (NL) receive input from both ipsilateral and contralateral nucleus magnocellularis (NM) neurons, with the axons of the latter acting as delay lines. A given neuron in the NL responds maximally to coinciding input from both NM neurons. To achieve maximum resolution of sound localization in the NL, the conduction velocity along these delay lines must be precisely tuned. Here, we examined the development of this velocity between embryonic days (E)12 and E18. Our optical imaging approach visualizes the contralateral delay lines along almost the complete NL of the chicken embryo. Optical imaging with the voltage-sensitive dye RH 795 showed no significant differences in the velocity between E12 and E15, but a significant increase from E15 to E18, at both 21 degrees C and 35 degrees C. Surprisingly, at 21 degrees C the conduction velocity in the dorso-lateral part of the NL was significantly higher compared to the situation in the ventro-medial part. The observed development in contralateral conduction velocity may be due to a developmental increase in myelination of the NM axons. Indeed, antibody staining against myelin-associated glycoprotein (alpha-MAG) showed no myelination of the NM axon branches within the NL at E12 and E15. On the other hand, a clear alpha-MAG immunoreactivity occurred at E18. Our results therefore describe the developmental physiological properties of the delay line in the chicken embryo.

Study of the reproductive effects in rats surgically implanted with depleted uranium for up to 90 days.

In 2001, the Naval Health Research Center Toxicology Detachment was funded by the U.S. Army Medical Research Acquisition Activity (USAMRAA) to conduct a study of the effects of surgically implanted depleted uranium (DU) pellets on adult rat reproductive success and development across two successive generations. This article presents some of the findings for the group of offspring from adult rats mated at 30 d post surgical implantation of DU pellets. Adult male and female Sprague-Dawley rats (P1 generation) were surgically implanted with 0, 4, 8, or 12 DU pellets (1 x 2 mm). The P1 generation was then cross-mated at 30 d post surgical implantation. Urine collected from P1 animals at 27 d post surgical implantation showed that DU was excreted in the urine of DU-implanted animals in a dose-dependent manner. DU surgical implantation did not have a negative impact on P1 reproductive success, survival, or body weight gain through post surgical implantation d 90. There were no statistically significant differences in F1 birth weight, survival, and litter size at postnatal day (PND) 0, 5, and 20. No gross physical abnormalities identified in the offspring were attributable to neonatal DU exposure. A series of neurodevelopment and immune function assessments were also conducted on F1 offspring. No group differences were observed that were related to parental DU exposure. Studies are ongoing on the impact of leaving DU embedded in soft tissue for 120 d on rat reproduction and subsequent offspring survival and development.

Functional glutamatergic and glycinergic inputs to several superior olivary nuclei of the rat revealed by optical imaging.

Superior olivary complex (SOC) neurons receive excitatory and inhibitory inputs from both ears. We determined the nature of such inputs to the main SOC nuclei with an optical imaging system. To do so, brainstem slices of postnatal (P) rats (P3-13) were treated with the fast voltage-sensitive dye RH795, and ipsilateral and contralateral SOC inputs were activated electrically. Optical signals, equivalent to membrane potential changes, were detected by a 464-photodiode array. The signals consisted mostly of two components which were identified as pre- and postsynaptic potentials in experiments with Ca2+-free solutions. They correlated with morphological structures, i.e. the presynaptic components were prominent in neuropil regions whereas the postsynaptic components dominated in somata regions. Postsynaptic components were distinguished pharmacologically with the glycine receptor blocker strychnine and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Concerning the lateral superior olive, we confirmed the known glutamatergic inputs from the ipsilateral side and the glycinergic inputs from the ipsilateral and contralateral sides. Furthermore, we identified a CNQX-sensitive input from the contralateral side. In the medial superior olive, we corroborated the glutamatergic and glycinergic inputs from the ipsilateral and contralateral sides. Both ipsi- and contralaterally, the glutamatergic input was more pronounced than the glycinergic input. In the superior paraolivary nucleus, we also identified ipsilateral and contralateral inputs. Besides the known glycinergic input from the contralateral side, we found a novel glycinergic input from the ipsilateral side and identified CNQX-sensitive inputs from the contralateral and ipsilateral sides. The latter was very weak and appeared only in 30% of the experiments. The data show the feasibility of identifying functional inputs to the SOC with voltage-sensitive dye recordings.

Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli.

The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.

Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation.

1. The inhibitory neurotransmitter glycine can elicit depolarizing responses in immature neurones. We investigated the changes in glycine responses and their ionic mechanism in developing neurones of the rat lateral superior olive (LSO), an auditory brainstem nucleus involved in sound localization. 2. Whole-cell and gramicidin perforated-patch recordings were performed from visually identified LSO neurones in brain slices and glycine was pressure applied for 3-100 ms to the soma. Glycine-evoked currents were reversibly blocked by strychnine. They were mostly monophasic, but biphasic responses occurred in approximately 30 % of P8-11 neurones in perforated-patch recordings. 3. In whole-cell recordings from P2-11 neurones, the reversal potential of glycine-evoked currents (EGly) was determined by the transmembranous Cl- gradient and corresponded closely to the Nernst potential for Cl-, regardless of age. This indicates that Cl- is the principle ion permeating glycine receptors, but is also consistent with a low relative (10-20 %) permeability for HCO3-. The Cl- gradient also determined the polarity and amplitude of glycine-evoked membrane potential changes. 4. Leaving the native intracellular [Cl-] undisturbed with gramicidin perforated-patch recordings, we found a highly significant, age-dependent change of EGly from -46.8 +/- 1.8 mV (P1-4, n = 28) to -67.6 +/- 3.3 mV (P5-8, n = 10) to -82.2 +/- 4.1 mV (P9-11, n = 18). The majority of P1-4 neurones were depolarized by glycine ( approximately 80 %) and spikes were evoked in approximately 30 %. In contrast, P9-11 neurones were hyperpolarized. 5. In perforated-patch recordings, EGly was influenced by the voltage protocol and the glycine application interval; it could be shifted in the positive and negative direction. For a given application interval, these shifts were always larger in P1-4 than in P8-11 neurones, pointing to less effective Cl- regulation mechanisms in younger neurones. 6. Furosemide (frusemide), a blocker of cation-Cl- cotransporters, reversibly shifted EGly in the negative direction in P2-4 neurones, yet in the positive direction in P8-10 neurones, suggesting the blockade of net inward and net outward Cl- transporters, respectively. 7. Taken together, age-dependent changes in active Cl- regulation are likely to cause the developmental shift from depolarizing to hyperpolarizing glycine responses. A high intracellular [Cl-] is generated in neonatal LSO neurones which decreases during maturation.

Developmental expression of the glycine transporter GLYT2 in the auditory system of rats suggests involvement in synapse maturation.

The synaptic action of many neurotransmitters is terminated by specific transporters that remove the molecules from the synaptic cleft and help to replenish the transmitter supply. Here, we have investigated the spatiotemporal distribution of the glycine transporter GLYT2 in the central auditory system of rats, where glycinergic synapses are abundant. In adult rats, GLYT2 immunoreactivity was found at all relay stations, except the auditory cortex. Many immunoreactive puncta surrounded the neuronal somata in the cochlear nuclear complex, the superior olivary complex, and the nuclei of the lateral lemniscus. In contrast, diffuse neuropil labeling was seen in the inferior colliculus and the medial geniculate body. The punctate perisomatic labeling and the diffuse neuropil labeling were very similar to the staining pattern described previously with glycine antibodies in the auditory system, suggesting that GLYT2 is a reliable marker for glycinergic synapses. However, there was a discrepancy between cytoplasmic GLYT2 and glycine labeling, as not all neuron types previously identified with glycine antibodies displayed somatic GLYT2 immunoreactivity. During development, GLYT2 immunoreactivity appeared between embryonic days 18 and 20, i.e., shortly after the time when the earliest functional synapses have been established in the auditory system. Labeling turned from a diffuse pattern to a clustered, punctate appearance. The development was also characterized by an increase of the signal intensity, which generally lasted until about postnatal day 10. Thereafter, a decrease occurred until about postnatal day 21, when the mature pattern was established in most nuclei. Because of the perinatal onset of GLYT2 immunoreactivity, we speculate that the transporter molecules participate in the process of early synapse maturation.

Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A. tumefaciens RpoA gene, encoding the alpha subunit of RNA polymerase.

The two-component regulatory system, composed of virA and virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However, virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the alpha subunit of RNA polymerase (RNAP), which confers significant expression of a virB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulator virG gene. We conducted in vitro transcription assays using either reconstituted E. coli RNAP or hybrid RNAP in which the alpha subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a sigma(70)-dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBp in a virG-dependent manner. In addition, we provide evidence that the alpha subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the alpha subunit of A. tumefaciens and that the alpha subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine vir gene expression as well as the T-DNA transfer process in E. coli.

Immunohistochemical localization of the somatostatin sst2(b) receptor splice variant in the rat central nervous system.

Somatostatin is a neuromodulator in the mammalian CNS. To date, genes for at least five different somatotrophin release inhibiting factor receptors, termed sst1-sst5, have been cloned. The rat sst2 receptor exists in two splice variants, sst(alpha)a) and sst2(b), which differ in their carboxy-termini. When heterologously expressed in Chinese hamster ovary-K1 cells, these splice variants show little difference in their operational characteristics. Recently, the distribution of the sst2(a) receptor was documented, yet at present no data are available about the distribution of the sst2(b) receptor in the CNS. Here, we present the characterization of a novel polyclonal anti-peptide antibody that is selective for the sst2(b) receptor splice variant. The antibody was raised against the unique intracellular carboxy-terminal portion of the receptor protein. Using this affinity-purified antibody in western blotting experiments, the sst2(b) receptor expressed in Chinese hamster ovary-K1 cells was shown to be a glycoprotein with a molecular weight centred at about 85,000. The antibody showed no cross-reactivity to any of the recombinant human sst1-5 receptors, the rat sst2(a) receptor or wild-type Chinese hamster ovary-K1 cells. Employing immunohistochemistry, we investigated the distribution of the sst2(b) receptor in the brain and spinal cord of adult rats. A distinct distribution was found throughout the rostrocaudal axis of the CNS. Somatodendritic as well as axonal staining was observed. Somatodendritic labelling was particularly obvious in the olfactory bulb, cerebral cortex, hippocampal formation, mesencephalic trigeminal nucleus and cerebellum, as well as in cranial and spinal motor areas. The results show that the distribution of the sst2(b) receptor partially overlaps with that of the sst2(b) receptor, although there were differences in a number of brain areas. The location of the sst2(b) receptor implies that it may mediate a modulatory role of somatostatin inhibitory releasing factor on sensory as well as motor functions.

Electrical membrane properties of trapezoid body neurons in the rat auditory brain stem are preserved in organotypic slice cultures.

The medial nucleus of the trapezoid body (MNTB) is a conspicuous structure in the mammalian auditory brain stem. It is a major component of the superior olivary complex and is involved in sound localization. Recently, organotypic slice culture preparations of the superior olivary complex were introduced to investigate the development of inhibitory and excitatory projections (Sanes and Hafidi, 1996; Lohmann et al., 1998). In the present article, we further assessed the organotypicity of our culture system (Lohmann et al., 1998) and examined electrical membrane properties of MNTB neurons expressed under culture conditions. To do so, MNTB neurons from early postnatal rats (P3-5) were studied after 3-6 days in vitro (DIV) by whole-cell patch-clamp recordings. Their mean resting potential was -59 mV, the input resistance averaged 171 Momega, and the average time constant was 3 ms. Four types of voltage-activated conductances were observed in voltage-clamp recordings. All cells expressed a tetrodotoxin (TTX)-sensitive sodium current. Two types of potassium currents could be characterized: a tetraethylammonium (TEA) -sensitive and a 4-aminopyridine (4-AP)-sensitive conductance, both of which are composed of a transient and a sustained component. Finally, an inwardly rectifying current, activated by hyperpolarizing voltage steps, was found. In current-clamp recordings, depolarizing current pulses typically elicited a single action potential. In the presence of 4-AP, however, these current pulses induced a train of action potentials. The duration of action potentials was increased by 4-AP and the afterhyperpolarization was reduced. Hyperpolarizing current injections induced a "sag" in the membrane potential, indicating the influence of an inwardly rectifying current. Our results demonstrate that MNTB neurons in slice cultures have electrical membrane properties comparable to those of their counterparts in acute slices.

The Bradyrhizobium japonicum noeD gene: a negatively acting, genotype-specific nodulation gene for soybean.

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.

Impact on arbuscular mycorrhiza formation of pseudomonas strains used as inoculants for biocontrol of soil-borne fungal plant pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 &mgr;M DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.

Kainate responses of leech Retzius neurons in situ and in vitro.

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5-20 microM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 microM kainate. In RCs in situ the membrane responses to 5 microM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 microM kainate were similar to the membrane response to 20 microM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca2+]i) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca2+]i, indicating that the Ca2+ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca2+]i occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca2+ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 microM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca2+ and high-Mg2+ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2-7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca2+ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified.

Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, in the mammalian retina.

Synaptophysin and synaptoporin are homologous proteins that are among the most abundant synaptic vesicle proteins. Despite their high degree of sequence similarity, they are differentially distributed in the brain. The distribution of synaptophysin and synaptoporin was examined in the adult rat and rabbit retina by using single- and double- labeling immunocytochemistry with conventional light microscopy and confocal laser scanning microscopy. In the rat retina, synaptophysin immunoreactivity was found in the outer plexiform layer in terminals of photoreceptors and was homogeneously distributed throughout the inner plexiform layer. Synaptoporin immunoreactivity, however, was restricted to the inner plexiform layer. Labeling was most prominent in three distinct bands of the inner plexiform layer separated by two bands of very low synaptoporin immunoreactivity. In the rabbit retina, synaptophysin and synaptoporin immunoreactivity were found in the inner and outer plexiform layers. In the inner plexiform layer, labeling for both vesicle proteins was homogeneous, with no detectable stratification. In the outer plexiform layer, synaptophysin was present in photoreceptor terminals, and synaptoporin was present in horizontal cells. Staining of isolated rabbit retinal cells confirmed that both the axonless A type and the axon-bearing B type horizontal cells are immunoreactive for synaptoporin. In addition, electron microscopy of synaptoporin-immunostained rabbit retinas revealed no labeling of photoreceptor terminals but of putative synaptic sites in horizontal cells in the outer plexiform layer. No functional correlation was found in the expression of either synaptic vesicle protein with the type of neuron or synapse (ribbon or conventional).

Host-Controlled Restriction of Nodulation by Bradyrhizobium japonicum Strains in Serogroup 110.

We previously reported the identification of a soybean plant introduction (PI) genotype, PI 417566, which restricts nodulation by Bradyrhizobium japonicum MN1-1c (USDA 430), strains in serogroup 129, and USDA 110 (P. B. Cregan, H. H. Keyser, and M. J. Sadowsky, Appl. Environ. Microbiol. 55:2532-2536, 1989, and Crop Sci. 29:307-312, 1989). In this study, we further characterized nodulation restriction by PI 417566. Twenty-four serogroup 110 isolates were tested for restricted nodulation on PI 417566. Of the 24 strains examined, 62.5% were restricted in nodulation by the PI genotype. The remainder of the serogroup 110 strains tested (37.5%), however, formed significant numbers of nodules on PI 417566, suggesting that host-controlled restriction of nodulation by members of serogroup 110 is strain dependent. Analysis of allelic variation at seven enzyme-encoding loci by multilocus enzyme electrophoresis indicated that the serogroup 110 isolates can be divided into two major groups. The majority of serogroup 110 isolates which nodulated PI 417566 belonged to the same multilocus enzyme electrophoresis group. B. japonicum USDA 110 and USDA 123 were used as coinoculants in competition-for-nodulation studies using PI 417566. Over 98% of the nodules formed on PI 417566 contained USDA 123, whereas less than 2% contained USDA 110. We also report the isolation of a Tn5 mutant of USDA 110 which has overcome nodulation restriction conditioned by PI 417566. This mutant, D4.2-5, contained a single Tn5 insertion and nodulated PI 417566 to an extent equal to that seen with the unrestricted strain USDA 123. The host range of D4.2-5 on soybean plants and other legumes was unchanged relative to that of USDA 110, except that the mutant nodulated Glycine max cv. Hill more efficiently. While strain USDA 110 has the ability to block nodulation by D4.2-5 on PI 417566, the nodulation-blocking phenomenon was not seen unless strain USDA 110 was inoculated at a 100-fold greater concentration than the mutant strain.

Expression of neurofilament proteins by horizontal cells in the rabbit retina varies with retinal location.

Classical neurofibrillar staining methods and immunocytochemistry with antibodies to the light, medium and heavy chain subunits of the neurofilament triplet have been used for in situ and in vitro investigation of the organization of neurofilaments in A- and B-type horizontal cells of the adult rabbit retina. Surprisingly, their expression and organization within a cell is dependent on its location along the dorso-ventral axis of the retina. A-type horizontal cells in superior retina consistently stained with a wide variety of neurofibrillar methods to reveal neurofibrillar bundles, which immunocytochemistry showed to contain all three neurofilament subunits. A-type horizontal cells in inferior retina were uniformly refractory to neurofibrillar staining, although they expressed all three subunits. However, there was less of the light and medium subunits; the organization of the filaments into bundles (neurofibrils) is minimal. B-type horizontal cells could not be stained with any neurofibrillar method and were not recognizable by in situ immunocytochemistry. However, B-type cells could be seen to express all three subunits in vitro, but the expression of the light and medium subunits was weak. There was only a slight difference between B-type cells taken from superior and inferior retina. Combined with the results of recent transfection studies, these findings suggest that the amount of the light neurofilament subunit present in a horizontal cell determines its content of neurofibrillar bundles, and that rabbit horizontal cells may contain more neurofilament protein, particularly of the heavy subunit, than is used for neurofilament formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity of neuronal phenotypes expressed in monolayer cultures from immature rabbit retina.

We have used monolayer cultures prepared from early postnatal rabbit retinae (days 2-5) by the sandwich technique to study the capacity of immature neurons to express specific neuronal phenotypes in a homogeneous in vitro environment. Applying morphological, immunocytochemical, and autoradiographic criteria, we demonstrate that a variety of phenotypes could be distinguished after 7-14 days in vitro, and correlated with known retinal cell types. Bipolar cell-like neurons (approximately 4% of total cell number) were identified by cell type-specific monoclonal antibodies (115A10) and their characteristic bipolar morphology. Small subpopulations (about 1%) of GABA-immunoreactive neurons acquired elaborate morphologies strikingly similar to those of A- and B-type horizontal cells. Amongst putative amacrine cells several different subpopulations could be classified. GABA-immunoreactive amacrine-like neurons (6.5%), which also showed high affinity [3H]-GABA uptake, comprised cells of varying size and shape and could be subdivided into subpopulations with respect to their response to different glutamate receptor agonists (NMDA, kainic acid, quisqualic acid). In addition, a small percentage of [3H]-GABA accumulating cells with large dendritic fields showed tyrosine-hydroxylase immunoreactivity. Presumptive glycinergic amacrine cells (18.5%) were rather uniform in shape and had small dendritic fields. Release of [3H]-glycine from these neurons was evoked by kainic and quisqualic acid but not by NMDA. Small [3H]-glutamate accumulating neurons with few short processes were the most frequent cell type (73%). This cell type also exhibited opsin immunoreactivity and probably represented incompletely differentiated photoreceptor cells. Summing the numbers of characterized cells indicated that we were able to attribute a defined retinal phenotype to most, if not all of the cultured neurons. Thus, we have demonstrated that immature neuronal cells growing in monolayer cultures, in the absence of a structured environment, are capable of maintaining or producing specific morphological and functional properties corresponding to those expressed in vivo. These results stress the importance of intrinsic factors for the regulation of neuronal differentiation. On the other hand, morphological differentiation was far from perfect indicating the requirement for regulatory factors.

Voltage-gated currents of rabbit A- and B-type horizontal cells in retinal monolayer cultures.

In monolayer cultures prepared from immature early postnatal rabbit retina, small populations of neurons can be demonstrated to differentiate into apparently mature A- and B-type horizontal cells. Using whole-cell, single-channel, patch-clamp recording techniques, we have analyzed the pattern of voltage-gated conductances expressed by mammalian horizontal cells under these conditions. A total of six different voltage-dependent ionic currents were recorded. Tetrodotoxin-sensitive fast sodium inward currents (INa) were found in 81% of the A-type and 90% of the B-type cells. Inward calcium currents could be demonstrated in all cells tested after blockade of other conductances. Two types of outward potassium currents with properties of the 4-aminopyridine-sensitive transient IA and the tetraethylammonium sensitive delayed rectifier IK, respectively, could be characterized in whole-cell recordings. An inward rectifying potassium current (Ianom) typical for horizontal cells was activated in response to hyperpolarizing voltage steps. These types of currents have also been described in dissociated adult horizontal cells from lower vertebrates and cat. With single-channel recordings on inside-out patches excised from B-type cells, an additional Ca(2+)-dependent current (IK(Ca)) was observed which, so far, has not been described in horizontal cells developing in situ. Our results demonstrate that cultured rabbit horizontal cells express a set of voltage-gated currents which largely, but not completely, corresponds to that described in situ for horizontal cells of other species. The culture system will allow further investigation of developmental and functional aspects of mammalian horizontal cells.

Expression of horizontal cell phenotypes in monolayer cultures from immature rabbit retina.

Using the sandwich culture technique introduced by Brewer and Cotman we have studied the in vitro differentiation of A- and B-type horizontal cells which represent two well characterized cell types of the rabbit retina. Neurons from immature (postnatal day 3) rabbit retinae were dissociated and grown on inverted coverslips for up to 5 weeks in a chemically defined medium. On the basis of morphological criteria and the staining pattern for several immunocytochemical and autoradiographic horizontal cell markers we have examined to what extent expression of a distinct mature neuronal phenotype can take place under the artificial conditions of monolayer cultures. After 14 days in vitro neurons could be identified which had acquired elaborate morphological features closely resembling those of A- and B-type horizontal cells, respectively. Axonless A-like cells had 2-4 stout primary dendrites. In agreement with in situ observations these cells showed immunoreactivity for neurofilament proteins (68 kDa, 200 kDa), calbindin-28 kDa and less strongly for vimentin. B-like neurons reached varying states of development. Ideally, they had dendritic trees with 6-8 primary processes extending radially from the soma and a single axon-like process which branched extensively to form a profuse neuritic arbor strikingly similar to axon terminal systems of B-type cells in the intact retina. B-like cells also stained for vimentin, calbindin-28 kDa and unexpectedly also for neurofilament proteins. Interestingly, however, neurofilaments became redistributed during in vitro development eventually resulting in their restricted localization in the 'axon terminal system'. This apparently reflects a developmental process which has escaped detection in situ so far. Both cell types were intensely labelled with antibodies to gamma-aminobutyric acid (GABA), the presumed horizontal cell transmitter, but high affinity uptake of this transmitter was practically undetectable by [3H]-GABA autoradiography. This was in agreement with observations in intact retinae. These results support the notion that once a neuron has reached a certain developmental state further differentiation and maintenance of its particular morphological and functional properties are primarily governed by intrinsic factors, but do not exclude that extrinsic signals have important modulatory functions.

Glucose stimulates cAMP accumulation in the oral bacterium Actinomyces viscosus.

Actinomyces viscosus T14V, a Gram-positive bacterium found in the oral cavity, was found to be insensitive to glucose-mediated catabolite repression. Basal levels of beta-galactosidase (18-26 U) were observed at all phases of growth regardless of the culture conditions. Further, beta-galactosidase could not be induced with lactose, or with a known inducer of the enzyme, isopropyl-beta-D-thiogalactoside, or with dibutyryl cAMP. Glucose, on the other hand, stimulated cAMP accumulation in a concentration-dependent manner. Fructose and sucrose mimicked the effects of glucose on cAMP accumulation, whereas galactose, mannose and maltose had lesser stimulatory effects. Other carbon sources, i.e., lactose, alpha-methylglucoside, ribose, xylose and succinate were without effect. Glucose and alpha-methylglucoside were found to stimulate cAMP accumulation in toluene-permeabilized cells, in the presence of the phosphodiesterase inhibitor, theophylline. Glucose did not stimulate cAMP levels in other Gram-positive bacteria including Streptococcus mutans, S. sanguis and S. salivarius but did cause cAMP accumulation in other strains of A. viscosus. The results suggest that glucose effects on cAMP metabolism are independent of the induction of beta-galactosidase as presently defined for Escherichia coli, and that the effects appear to be selective to the A. viscosus bacteria. The results also suggest that glucose stimulates cAMP accumulation via activation of adenylate cyclase.

Inability to express fimbriae results in impaired ability of Haemophilus influenzae b to colonize the nasopharynx.

We cloned into the structural fimbrial subunit gene from a fimbriated Haemophilus influenzae b a 1.5-kb kanamycin resistance gene. The resultant strain (RKAW5) was tested by Southern analysis, hemagglutination, and electron-micrographic examination to confirm gene inactivation. In comparison with the parent, RKAW5 exhibited a significant decrease in adherence to human buccal epithelial cells and in nasal colonization of yearling rhesus monkeys.