Reproductive morphology and its application in testing molecular systematic hypotheses in the family Gobiidae (Teleostei, Gobiiformes).

This study uses histological techniques to make a detailed comparison of the reproductive morphologies of four gobiid genera, Amblyeleotris, Ctenogobiops, Fusigobius and Kraemeria. Three distinct reproductive morphological patterns were observed. All species examined in the genus Fusigobius exhibit either an ovariform or testiform gonad and precursive accessory gonadal structures (pAGS) associated with each of the gonadal lobes, regardless of gonadal state. In contrast, among species of Amblyeleotris, Ctenogobiops and Kraemeria examined, pAGS were not found. Furthermore, Amblyeleotris and Ctenogobiops differ from both Kraemeria and Fusigobius in lacking AGS associated with the testiform gonad. These findings, based solely on reproductive morphology, suggest that Kraemeria and Fusigobius may be more closely related to each other than either is to Amblyeleotris and Ctenogobiops. Findings of this study support the view that reproductive morphological patterns could prove informative in elucidating evolutionary relationships within the family Gobiidae.

Rediscovering hermaphroditism in Grammatidae with the description of the testicular gland in Brazilian Basslet Gramma brasiliensis.

Many aspects of sex change in reef fishes have been studied, including behavior and social organization. However, gonad histology remains the most robust way to identify sexual patterns in fishes. Some uncommon tissues remain poorly described, such as the accessory gonadal structures found in species from the Gobiidae family, which are rare in other bony fishes. This is the first report of the testicular gland in Gramma brasiliensis and for the Grammatidae family. Between April 2011 and February 2012 eighty specimens were collected during four dive campaigns on the Taipus de Fora reef (13°56'20"S 38°55'32"W), Bahia, Northeast Brazil, and their sex was determined. Thirteen per cent of the active-females and 90% of the active-males had testicular gland tissue in their ovotestis. This discovery led to additional research into the characteristics of the gland tissue and its relationship with gonadal maturation. Three patterns of testicular gland development were found in Brazilian basslet ovotestis. Both ova and sperm-producing gonad contained testicular gland tissue, and the appearance of this tissue seems to be the first modification of ovotestis tissue marking the beginning of the protogynous sex-change process in G. brasiliensis.

Resistivity of axoplasm. I. Resistivity of extruded squid axoplasm.

Six methods have given squid axoplasm resistivities of from 1.0 to 6.9 times seawater (X SW), so another was tried. A 100-mum platinized electrode was to be inserted from each end of an axion in iso-osmotic sucrose and impedance between them measured vs. separation. But observations that the resistance of axons in sucrose increased steadily ruled this out. Axoplasm from two or three axons was transferred to a glass capillary, 0.6 mm ID, and the 1-kHz series resistance and reactance were measured at electrode separations from 16 to 2 mm. The resistance was linear vs. distance, giving the resistivity, while the reactance was nearly constant, implying constant electrode contributions. Frequency runs from 10 Hz to 30 kHz at 10 mm gave electrode impedances of the form (jomega)-alpha, allowing 1-2% effects on the axoplasm resistivities. In nine experiments, one was discarded for cause, the range and average resistivities were, respectively, 1.2-1.6 and 1.4 times those of artificial seawater (19.7 omegacm at 24.4 degrees C). No single cause for the variability was apparent. These experiments essentially confirm the means and variations of two early experiments with intact axons and recent results with a single internal electrode to give overall resistivities of 1.4 +/- 0.2 X SW.

Descending projections from auditory brainstem nuclei to the cochlea and cochlear nucleus of the guinea pig.

Projections from auditory brainstem nuclei to the cochlea and cochlear nuclei in the guinea pig were studied by injection of two retrograde fluorescent neuronal tracers. For seven experiments fast blue was injected into the scala tympani of one cochlea and diamidino yellow was injected into dorsal or anteroventral cochlear nucleus of the same side. The results show that the efferent projections to the cochlea and cochlear nucleus generally form two separate neuronal systems even though they share many common nuclei of origin. The largest projections to the cochlear nucleus come bilaterally from the lateral and ventral nuclei of the trapezoid body. Other nuclei, the lateral superior olive, the ventral nucleus of the lateral lemniscus, the dorsomedial periolivary nuclei, and the medial nucleus of the trapezoid body showed an ipsilateral bias in their projections to the cochlear nucleus. An upper limit of 3.5% of the medial system olivocochlear efferent neurones projecting to the cochlea were labelled with both diamidino yellow and fast blue, suggesting that few efferent neurones projecting to the cochlea send collaterals to the cochlear nucleus in this species. However, the site of medial system olivocochlear efferent collateral terminations is the granule cell area for the cat, mouse, and gerbil. When diamidino yellow was injected in the superficial layers of the cochlear nucleus, including the superficial granule cell layer of the ventral cochlear nucleus, approximately 3.6% of medial system olivocochlear efferents projecting to the cochlea sent collaterals to the cochlear nucleus. In three animals fast blue was injected into the cochlear nucleus and diamidino yellow into the cochlea. These experiments revealed a greater proportion of the medial system olivocochlear efferents projecting to the cochlea sending collaterals to the cochlear nucleus, but this proportion was still less than 10%. These results were confirmed by the extracellular injection of horseradish peroxidase into the intraganglionic spiral bundle. Only three medial system olivocochlear efferents were observed to send collaterals to the cochlear nucleus. This number was less than 10% of all labelled medial system fibres. Although these experiments suggest that in the guinea pig the number of olivocochlear efferents sending collaterals to the cochlear nucleus is considerably smaller than is found for the cat, mouse, and gerbil, it is not possible with the current experimental procedures to conclude whether the results are due to species or methodological differences.

Local mu and delta opioid receptors regulate amphetamine-induced behavior and neuropeptide mRNA in the striatum.

The purpose of this study was to investigate the role that mu and delta opioid receptor blockade has upon stimulant-induced behavior and neuropeptide gene expression in the striatum. Acute administration of amphetamine (2.5 mg/kg i.p.) caused an increase in behavioral activity and preprodynorphin, substance P, and preproenkephalin mRNA expression. Intrastriatal infusion of the mu opioid antagonist, H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), or the delta opioid antagonist, H-Tyr-Tic[CH(2)NH]-Phe-Phe-OH (TIPPpsi), significantly decreased amphetamine-induced vertical activity. However, only CTAP reduced amphetamine-induced distance traveled. Quantitative in situ hybridization histochemistry revealed that CTAP blocked amphetamine-induced preprodynorphin and substance P mRNA. However, preproenkephalin mRNA levels in the dorsal striatum were increased to the same extent by CTAP, amphetamine, or a combination of the two drugs. In contrast, TIPPpsi significantly decreased amphetamine-induced mRNA expression of all three neuropeptides. These data indicate that both mu and delta receptor subtypes differentially regulate amphetamine-induced behavior and neuropeptide gene expression in the rat striatum.

Maturation of immune responses to lentivirus infection: implications for AIDS vaccine development.

The evaluation of attenuated vaccines in the simian immunodeficiency virus and equine infectious anemia virus animal models has demonstrated the ability of this immunization strategy to elicit broad and enduring immune protection from virus exposure. The development of protective immunity by these attenuated virus vaccines, however, has been shown to be time dependent and to be associated with a complex and lengthy maturation of immune responses over the first 6 to 8 months postinoculation. During this time period, envelope-specific antibody responses undergo an evolution in quantitative and qualitative properties that is similar, but distinct for each lentivirus system. The completed maturation of immune responses is then characterized by relatively steady-state antibody responses that are maintained indefinitely. The accomplishment of optimum vaccine protection is associated with achievement of a fully mature immune response, whereas nonprotective or enhancing vaccine immunity appears to be associated with immature immune responses elicited by ineffective vaccines. These observations indicate that the development of an effective acquired immunodeficiency syndrome (AIDS) vaccine will require immunization strategies that can achieve the necessary maturation of immune responses to human immunodeficiency virus type 1 (HIV-1) antigens in the minimum amount of time. Therefore, AIDS vaccine strategies based on attenuated live virus vaccines or on DNA immunization procedures, perhaps in conjunction with cytokine or secondary costimulatory molecules to accelerate immune maturation, may be best suited to accomplish the goal of an effective and practical AIDS vaccine for worldwide use.

Production and characterization of SIV envelope-specific rhesus monoclonal antibodies from a macaque asymptomatically infected with a live SIV vaccine.

Five rhesus monoclonal antibodies (RhMAbs) were produced by rhesus EBV transformation of peripheral blood B cells from a rhesus macaque that had been asymptomatically infected with an attenuated, macrophage-tropic SIV strain, 17E-Cl. These MAbs recognized conformation-dependent epitopes on SIV gp120 and could not be mapped using synthetic peptides. All five RhMAbs were able to neutralize the vaccine strain and a heterologous isolate, SIV/DeltaB670. The RhMAbs did not cross-react with HIV-2; by contrast, four human MAbs derived from an HIV-2-infected person were broadly cross-reactive with both SIV and HIV-2 gp120s. Cross-competition analysis indicated that the five RhMAbs could be placed in two groups recognizing two nonoverlapping epitopes; while the HMAbs were placed in two additional competition groups. Binding of the three group I RhMAbs (1.7F, 3.11B, and 1.10A) as well as HMAb 17A was shown to be sensitive to specific amino acid alterations in V4 occurring in natural env variants. The results of this study demonstrate that RhEBV transformation provides a means to probe rhesus antibody responses to SIV infection at the monoclonal level. RhMAbs will allow structural and functional studies of envelope glycoprotein determinants that elicit protective immune responses against SIV.

Early efferent innervation of the developing rat cochlea studied with a carbocyanine dye.

The olivocochlear pathway in the developing rat was visualized in fixed material. The fluorescent carbocyanine dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to the cut central axons of the olivocochlear neurones at the floor of the fourth ventricle, and the termination pattern within the cochlea was examined after dye diffusion. From the day of birth (P0) to postnatal day 2 (P2), efferent innervation of the cochlea was exclusively in the region of the inner hair cells. Between P2 and P11, progressive outgrowth of neuronal processes to the outer hair cell region occurred; possible connections with the outer hair cells were occasionally seen at P4 and approached the mature pattern by P6. The efferent innervation of the organ of Corti appeared to mature progressively from the cochlear base to the apex, with outgrowth to the outer hair cells occurring earlier in the basal turn of the cochlea than in the second and third cochlear turns. Numerous blind axonal endings were observed in the spiral lamina especially at early postnatal ages. These findings may be consistent with a sequential pattern of arrival of efferent axons at the organ of Corti and ongoing death of efferent neurones in the brainstem during this period of development.

Postnatal development of the efferent innervation of the rat cochlea.

The postnatal development of the efferent innervation of the rat cochlea was studied by intracochlear injection of the fluorescent retrograde neuronal traces Diamidino yellow and Fast blue. Injections were performed on adult rats and on neonatal rats ranging from 0 to 8 postnatal days. It was found that the total number of neurones labelled in the brainstem after intracochlear injection was not significantly different in the newborn rat, compared to the adult. On the basis of cell body location and laterality of projections, there was a clear separation into lateral and medial efferent systems at the earliest postnatal age studied (PO). Evidence was also found in the newborn for a tonotopicity in the lateral system projection similar to that in the adult. Differences between the newborn and adult were a slight but significantly greater number of bilaterally-projecting cells in the newborn, and the presence in the newborn of a small number of cells located in the lateral superior olivary nucleus contralateral to their target cochlea. These were extremely rare in the adult brainstem. Evidence was found for the occurrence of postnatal neuronal death in nuclei of origin of both efferent systems. It is suggested that although the overall extent and general organization of the efferent projection to the cochlea in the rat appears to be established at birth, regressive changes are occurring during the postnatal shaping and maturation of this brainstem-to-cochlea pathway.

Effect of spider venom on cochlear nerve activity consistent with glutamatergic transmission at hair cell-afferent dendrite synapse.

Venom from the spider Argiope trifasciata, a highly specific blocker of the ionic channels associated with invertebrate glutamatergic receptors, was perfused through scala tympani of the basal turn of the pig cochlea. Its effect on spontaneous and driven activity of single afferent neurons was studied. 0.1 U/ml spider venom altered the maximum driven activity without an effect on spontaneous activity. 1 U/ml spider venom suppressed both spontaneous and driven activity. These findings are consistent with the hypothesis that L-glutamate is the neurotransmitter of the synapse between inner hair cells and primary auditory afferent neurons. The results also suggest that the differences in spontaneous activity between neurons may originate in variations in neurotransmitter release.

Quantitative estimate of bilaterally projecting medial olivocochlear neurones in the guinea pig brainstem.

Bilateral intracochlear injections of the fluorescent retrograde neuronal tracers fast blue and diamidino yellow were used to identify efferent neurones in the guinea pig brainstem which project to the inner ear on both sides. Bilaterally projecting neurones, detected by their double-labelled appearance in fluorescence microscopy, were found in all the nuclei of origin of the large neurone extra-LSO system of olivocochlear neurones. No double-labelled neurones were found in the small neurone LSO system. When precautions were taken to maximize their capture, these bilaterally projecting neurones comprised 5.4% of the total extra-LSO system neurones projecting to any one cochlea. On the basis of these quantitative data, it was suggested that the bilaterally projecting neurones may correspond to the small group of binaurally activated cochlear efferents identified in physiological studies.

Segregation of efferent projections to different turns of the guinea pig cochlea.

Localized intracochlear injections of the fluorescent retrograde label diamidino yellow were used to investigate the organization of efferent projections from the brainstem to different turns of the cochlea, in the guinea pig. It was found that the location of small neurones within the lateral superior olive ipsilateral to the injection varied in a systematic manner when injections proceeded from base to apex of the cochlea. In addition, a cruder form of cochleotopic organization was present in that most of the large medial system efferent neurones were labelled only after injection into the 3 most basal turns of the cochlea. The decline of medial system efferent innervation proceeding from base to apex was most striking for the contralateral efferent neurones. The details of base to apex innervation density were different for the different nuclei of origin of the medial system, implying the existence of complex subsystems within the medial neurone population.

Development of early social behaviour of rainbow trout, Salmo Gairdneri (Pisces, Salmonidae).

Young rainbow trout do not begin agonistic behaviour until they are free-swimming. Agonistic behaviour initially consists mostly of simple, straightforward attacks. More complex threat patterns are added with increasing age. Increased water flow rate promotes station-holding and apparently territorial behaviour, especially at low fish densities. High fish densities lead to an increase in agonistic interactions, especially in fast flowing water. The interplay of these factors with the developmental processes of social behaviour is discussed.

Rediscovering hermaphroditism in Grammatidae with the description of the testicular gland in Brazilian Basslet Gramma brasiliensis / Redescobrindo o hermafroditismo em Grammatidae com a descrição da glândula testicular de Gramma brasiliensis

Abstract Many aspects of sex change in reef fishes have been studied, including behavior and social organization. However, gonad histology remains the most robust way to identify sexual patterns in fishes. Some uncommon tissues remain poorly described, such as the accessory gonadal structures found in species from the Gobiidae family, which are rare in other bony fishes. This is the first report of the testicular gland in Gramma brasiliensis and for the Grammatidae family. Between April 2011 and February 2012 eighty specimens were collected during four dive campaigns on the Taipus de Fora reef (13°56’20”S 38°55’32”W), Bahia, Northeast Brazil, and their sex was determined. Thirteen per cent of the active-females and 90% of the active-males had testicular gland tissue in their ovotestis. This discovery led to additional research into the characteristics of the gland tissue and its relationship with gonadal maturation. Three patterns of testicular gland development were found in Brazilian basslet ovotestis. Both ova and sperm-producing gonad contained testicular gland tissue, and the appearance of this tissue seems to be the first modification of ovotestis tissue marking the beginning of the protogynous sex-change process in G. brasiliensis.

Resumo Diversos aspectos da troca de sexo em peixes recifais vem sendo estudados, incluindo comportamentos e organização social. Entretanto, a histologia das gônadas continua sendo a maneira mais robusta para se identificar padrões sexuais em peixes. Alguns tecidos incomuns, tais como as estruturas anexas a gônada encontradas em espécies da família Gobiidae e raras em outras espécies são pouco estudados. Este trabalho é a primeira descrição da glândula testicular em Gramma brasiliensis e para a família Grammatidae. Entre abril de 2011 e fevereiro de 2012, oitenta espécimes foram coletados durante quatro amostragens no recife de Taipus de Fora (13°56’20”S 38°55’32”W), Bahia, Brasil, e tiveram seus sexos determinados. Treze por cento das fêmeas ativas e noventa por cento dos machos ativos apresentaram tecido da glândula testicular em suas gônadas. Esta descoberta levou ao estudo da características dessa estrutura e sua relação com a maturação gonadal. Foram identificados três padrões de desenvolvimento da glândula testicular nas gônadas do Gramma brasiliensis. Tanto as gônadas produtoras de espermatozoides quanto as de oócitos apresentaram tecido da glândula testicular, e o surgimento desse tecido parece ser a primeira modificação gonadal do início da troca de sexo protogínica em G. brasiliensis.

Dynamic evolution of antibody populations in a rhesus macaque infected with attenuated simian immunodeficiency virus identified by surface plasmon resonance.

BACKGROUND: Increasing evidence suggests that an effective AIDS vaccine will need to elicit broadly neutralizing antibody responses. However, the mechanisms of antibody-mediated neutralization have not been defined. Previous studies from our lab have identified significant differences in the rates of antibody binding to trimeric SIV envelope proteins that correlate with neutralization sensitivity. Importantly, these results demonstrate differences in monoclonal antibody (MAb) binding to neutralization-sensitive and neutralization-resistant envelope proteins, suggesting that one mechanism for virus neutralization may be related to the stability of antibody binding. To date, little has been done to evaluate the binding properties of polyclonal serum antibodies elicited by SIV infection or vaccination. METHODS: In the current study, we translate these findings with MAbs to study antibody binding properties of polyclonal serum antibody responses generated in rhesus macaques infected with attenuated SIV. Quantitative and qualitative binding properties of well-characterized longitudinal serum samples to trimeric, recombinant SIV gp140 envelope proteins were analyzed using surface plasmon resonance (SPR) technology (Biacore). RESULTS: Results from these studies identified two antibody populations in most of the samples analyzed; one antibody population exhibited fast association/dissociation rates (unstable) while the other population demonstrated slower association/dissociation rates (stable). Over time, the percentage of the total binding response of each antibody population evolved, demonstrating a dynamic evolution of the antibody response that was consistent with the maturation of antibody responses defined using our standard panel of serological assays. However, the current studies provided a higher resolution analysis of polyclonal antibody binding properties, particularly with respect to the early time-points post-infection (PI), that is not possible with standard serological assays. More importantly, the increased stability of the antibody population with time PI corresponded with potent neutralization of homologous SIV in vitro. CONCLUSIONS: These results suggest that the stability of the antibody-envelope interaction may be an important mechanism of serum antibody virus neutralization. In addition, measurements of the 'apparent' rates of association and dissociation may offer unique numerical descriptors to characterize the level of antibody maturation achieved by candidate vaccine strategies capable of eliciting broadly neutralizing antibody responses.

Electrical properties of the squid axon sheath.

The first impedance data on the squid giant axon gave a lossy membrane capacity of 1 muF/cm2 from 1-200 kHz. The remaining observations of one experiment up to 5 MHz were not included in the simple conclusions and have now been ascribed to the mostly Schwann cell sheath. The results of these fragmentary data, 1.6 omega-cm2 and 12.5 membrane layers, are within the range of recent current clamp and electron microscope conclusions to give them independent confirmation and support.

Analogue solution for electrical capacity of membrane-covered cubes in cubic array at high concentration.

Earlier measurements have shown that the equations derived for the resistance of suspension of spheres apply up to a volume concentration of 100% for close-packing forms. They have also shown, by a resistance-capacitance analogue, that they apply for the capacity of a close-packing of membrane-covered square cylinders in square array, approximating muscle and nerve. The present work is an extension of the two-dimensional to a three-dimensional array of membrane-covered cubes in a cubic array. It is found by measurements of a three-dimensional electrolytic analogue that the capacitance is indeed expressed by an extension of the analytic solution at low concentrations up to 100% volume concentration of the membrane-covered form, such as epithelial tissues. There is thus at least one example each of two-and three-dimensional forms which conform to the low concentration analysis up to 100% volume and so give a basis for the extensions to other and more complicated forms to complete a survey to work begun by Fricke in 1923.

Analogue solution for electrical capacity of membrane covered square cylinders in square array at high concentration.

Analytical solutions of Laplace equations have given the electrical characteristics of membranes and interiors of spherical, ellipsoidal, and cylindrical cells in suspensions and tissues from impedance measurements, but the underlying assumptions may be invalid above 50% volume concentrations. However, resistance measurements on several nonconducting, close-packing forms in two and three dimensions closely predicted volume concentrations up to 100% by equations derived from Maxwell and Rayleigh. Calculations of membrane capacities of cells in suspensions and tissues from extensions of theory, as developed by Fricke and by Cole, have been useful but of unknown validity at high concentrations. A resistor analogue has been used to solve the finite difference approximation to the Laplace equation for the resistance and capacity of a square array of square cylindrical cells with surface capacity. An 11 x 11 array of resistors, simulating a quarter of the unit structure, was separated into intra- and extra-cellular regions by rows of capacitors corresponding to surface membrane areas from 3 x 3 to 11 x 11 or 7.5% to 100%. The extended Rayleigh equation predicted the cell concentrations and membrane capacities to within a few percent from boundary resistance and capacity measurements at low frequencies. This single example suggests that analytical solutions for other, similar two- and three-dimensional problems may be approximated up to near 100% concentrations and that there may be analytical justifications for such analogue solutions of Laplace equations.