Afferent input is necessary for seasonal growth and maintenance of adult avian song control circuits.

The neural circuits that regulate song behavior in adult songbirds undergo pronounced seasonal changes in morphology, primarily in response to changes in plasma testosterone (T). Most song nuclei have T receptors. We asked whether seasonal growth and maintenance of nuclei within these circuits are direct responses to the effects of T or its metabolites or are mediated indirectly via the effects of T on afferent nuclei. Photosensitive white-crowned sparrows were exposed to one of three treatments. (1) The neostriatal nucleus HVc (also known as the "high vocal center") was lesioned unilaterally, and the birds were exposed to long-day (LD) photoperiods and breeding levels of T for 30 d. (2) Birds were exposed to LD plus T (LD+T) for 30 d; then HVc was lesioned, and the birds were killed after an additional 30 d exposure to LD+T. (3) HVc was lesioned, and the sparrows were housed on short-day (SD) photoperiods in the absence of T treatment for 30 d. In both LD+T groups, the direct efferent targets of HVc, the robust nucleus of the archistriatum (RA) and area X, were smaller ipsilateral to the lesion. The lesion did not prevent growth of the hypoglossal motor nucleus, which does not receive direct afferent input from HVc. RA and area X were also smaller ipsilateral to the lesion in the SD birds. These results indicate that afferent input is required both for the growth of adult song circuits in response to typical breeding photoperiod and hormone conditions and for the maintenance of efferent nuclei in either their regressed or enlarged states.

Activation-dependent regulation of galanin gene expression in gonadotropin-releasing hormone neurons in the female rat.

In rats, galanin is colocalized in GnRH neurons, and galanin mRNA in GnRH neurons is increased coincidentally with the preovulatory gonadotropin surge. Whether the induction of galanin mRNA in GnRH neurons at proestrus reflects the action of sex steroids is unknown. We tested this hypothesis by challenging ovariectomized rats (n = 7) with estrogen and progesterone (E/P) to induce a LH surge and measuring galanin mRNA in GnRH neurons to determine whether there was an associated induction of galanin message in these cells. We used single and double label in situ hybridization and image analysis to compare among groups the levels of both galanin mRNA and GnRH mRNA in GnRH neurons. We found that steroid-primed animals showed an approximately 400% induction of galanin mRNA signal in GnRH neurons over that in vehicle-treated animals. Second, we hypothesized that steroid-dependent events which induce the expression of galanin mRNA in GnRH neurons depend on transsynaptic input to GnRH neurons. We tested this hypothesis by examining the effect of a pharmacological blockade of the steroid-induced activation of GnRH neurons on levels of galanin mRNA in these cells. We killed groups of ovariectomized adult female rats at the peak of a E/P-primed LH surge (n = 7) and after steroid priming followed by blockade of the LH surge with either the general anesthetic pentobarbital (n = 7) or the specific alpha-adrenergic receptor blocker phenoxybenzamine (n = 7). When we examined signal levels representing galanin mRNA content in GnRH neurons, we observed a 4-fold increase in signal for galanin mRNA in the GnRH neurons of steroid-primed (E/P surge) animals compared with that in oil-treated controls (P < 0.0004). This increase in galanin mRNA was prevented when the LH surge was blocked by treatment with either pentobarbital or phenoxybenzamine (P < 0.03 and P < 0.0001 vs. E/P surge controls, respectively). Cellular levels of GnRH mRNA were not different among control, E/P, and E/P plus pentobarbital groups (P > 0.2). These observations suggest that an increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a LH surge. By inference, induction of galanin mRNA in GnRH neurons reflects their activation, possibly via afferent neurons that transduce the steroid signal to GnRH neurons.

Diurnal rhythm in proopiomelanocortin mRNA in the arcuate nucleus of the male rat.

We tested the hypotheses that in the male rat, expression of proopiomelanocortin (POMC) mRNA in cells of the arcuate nucleus displays a diurnal fluctuation and that expression of this rhythm is dependent upon the secretory products of the testis. To accomplish this, we sacrificed groups of testes-intact and castrated adult male rats throughout the day and compared levels of POMC mRNA in individual cells of the arcuate nucleus across time and between groups. Adult male rats were housed on a 12-12 L D cycle with lights on a 0600 h and were divided into groups that were either castrated or left intact. Four days later, pairs from these groups were sacrificed at 0600 h, 1200 h, 1800 h, 2400 h, and again at 0600 h (n = 4 per group at each time point). We used in situ hybridization and a computerized image analysis system to measure cellular levels of POMC mRNA, as reflected by the number of autoradiographic grains over individual cells in the rostral quarter of the arcuate nucleus (counting approximately 30 cells per animal). Using cosinor analysis, we observed that in intact male rats, POMC mRNA levels varied significantly over the 24 h day with a nadir value at 1800 h. In contrast, there was no significant diurnal variation in POMC mRNA levels in castrated animals. POMC mRNA levels were significantly greater in the intact compared with castrated animals at every time point (P < 0.01), except at 1800 h, when the groups did not differ significantly from one another.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain space for learned song in birds develops independently of song learning.

In numerous species of birds, individuals or species that sing larger numbers of song types have larger song control nuclei in their brains. The direction of the cause and effect relationship between the complexity of song behavior and brain space is unknown, however. The hypothesis that birds that learn large song repertoires develop large song nuclei was therefore tested with a songbird, the marsh wren (Cistothorus palustris). Males were hand-reared and tutored in the laboratory with either small (n = 8 males heard 5 song types) or large (n = 8 males heard 45 song types) repertoires. When the birds were adults, the number of song types each male sang was first determined, and then the volume and certain cellular attributes of the song nuclei HVC and RA were measured. The two groups of wrens showed large behavioral differences in the size of their learned song repertoires, but did not differ in either the volumes of HVC and RA or in neuronal size, number, or density within these nuclei. These results suggest that the relationship between behavioral song complexity and brain space found in this and other species develops largely independently of early song learning experience and the later production of those songs.

Fimbriation of Pseudomonas cepacia.

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA+, while another, PC109, was HA-. A fimbriated (f+) HA+ derivative of PC109 (PC2(13)) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 +/- 1.36 nm in width and 200 to greater than 2,100 nm in length (PCE2(13)) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE2(13) indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE2(13) reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV-like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE2(13) (f+). PCE2(13) had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.

Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes.

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Lesions of the anterior forebrain song control pathway in female canaries affect song perception in an operant task.

We tested whether the avian anterior forebrain pathway functions in song perception in female canaries, and whether it is specialized for conspecific song perception or functions more generally in auditory perception. Using operant conditioning methods, we trained female canaries to discriminate among synthetic sound stimuli, canary songs, and song sparrow songs. We also trained each bird to discriminate among visual stimuli to test for general effects of lesions on performance. When canaries had learned the discrimination tasks, bilateral electrolytic lesions of the lateral portion of the magnocellular nucleus of the anterior neostriatum (lMAN) were made. The lesioned birds were then tested on the previously learned discrimination tasks. Lesions that destroyed most or all of lMAN decreased the ability of female canaries to discriminate between previously learned pairs of acoustic stimuli of all types, while visual discrimination was unaffected. These results suggest that the female canary anterior forebrain pathway contributes to the perception of acoustic stimuli, with this contribution including heterospecific song and other acoustic stimuli as well as canary song.

Seasonal plasticity of the song control system in wild Nuttall's white-crowned sparrows.

Seasonal plasticity in the morphology of telencephalic nuclei that control song behavior has been reported for diverse species of songbirds. The only published report of a lack of seasonal changes in the song nuclei of a seasonally breeding bird is that of Baker et al. in the Nuttall's subspecies of white-crowned sparrow (Zonotrichia leucophrys nuttalli). In this study, they brought wild birds into the laboratory and exposed them to either "summer" or "winter" photoperiods. Previous studies have shown that exposing wild-caught white-crowned sparrows to long-day photoperiods in the laboratory may not induce circulating concentrations of testosterone (T) as high as those seen in wild breeding birds. Changes in circulating T are primarily responsible for the seasonal morphological changes in the song nuclei. To determine whether there is seasonal plasticity of the song system in this subspecies, we measured circulating T, morphological attributes of the song nuclei, and song behavior in wild Nuttall's white-crowned sparrows during the spring and fall. Testis size and circulating T concentrations were greater in spring than fall birds. The absolute volumes of the song nuclei HVc, RA, and Area X, and their volumes relative to those of either the total telencephalon or three thalamic nonsong nuclei, were significantly greater in the spring than fall sparrows. Song behavior also changed seasonally; fall birds sang shorter songs than did spring birds. These results show that there is seasonal plasticity of the song system in wild Nuttall's white-crowned sparrows. Seasonal plasticity can now be regarded as a common feature of the seasonally breeding songbirds studied thus far.

Simultaneous visualization of two cellular mRNA species in individual neurons by use of a new double in Situ hybridization method.

We present a new method for the simultaneous detection of two mRNA species within individual neurons. The technique involves the use of radio-labeled and digoxigenin-labeled cRNA probes, the application of which confers a high specificity and sensitivity to the in situ hybridization analysis. We demonstrate the use of this method by illustrating the coexpression of preprogonadotropin-releasing hormone (GnRH) mRNA and preprogalanin mRNA in neurons in the rat forebrain and report a distinct sexual dimorphism in galanin gene expression by GnRH neurons. Coupling this technology with semi-quantitative analysis of the mRNA species hybridized with the isotopically labeled mRNA would permit studies of gene regulation in individual cells among the heterogeneous populations of the brain.

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.

Predictive value of oropharyngeal cultures for identifying lower airway bacteria in cystic fibrosis patients.

Identifying lower respiratory pathogens in young, non expectorating cystic fibrosis (CF) patients has been problematic. Bronchial secretions are difficult to obtain, and little is known about lower airway flora in these patients. We collected simultaneous bronchial and oropharyngeal specimens in 43 CF patients in optimal respiratory status, including both expectorating (17) and nonexpectorating (26) patients, to determine the predictive value of oropharyngeal cultures for identifying lower airway pathogens. An additional goal was to characterize the lower respiratory flora of these patients. Predictive values were defined as the proportion of oropharyngeal culture results that accurately reflected the results of bronchial cultures. Predictive values of positive oropharyngeal cultures in nonexpectorating patients were 83% (95% confidence interval 36 to 100%) for Pseudomonas aeruginosa and 91% (59 to 100%) for Staphylococcus aureus. Predictive values of negative oropharyngeal cultures were lower: 70% (48 to 86%) for R aeruginosa and 80% (52 to 96%) for S. aureus. A relatively high proportion of nonexpectorating CF patients less than 10 yr old had R aerusginosa (11 of 24, 46%) or Klebsiella species (5 of 24, 21%) in their lower airways. The isolation of Klebsiella was associated with younger age (p = 0.03) and recent administration of antistaphylococcal antibiotics (p = 0.05). Our results suggest that oropharyngeal cultures yielding R aeruginosa or S. aureus are highly predictive, but such cultures lacking these organisms do not rule out the presence of these pathogens in the lower airways of CF patients.

Genetic analysis of a breast-ovarian cancer family, with 7 cases of colorectal cancer linked to BRCA1, fails to support a role for BRCA1 in colorectal tumorigenesis.

Mutations in the BRCA1 gene cause strongly elevated risks of breast and ovarian cancers but may also confer a 3-fold increased risk for colorectal cancer. To address the relationship between BRCA1 carriership and colorectal tumorigenesis, we studied the genetics of a breast-ovarian cancer family with 7 cases of colorectal cancer. A germline 3938insG mutation in BRCA1 was found in 5 breast-cancer patients, 1 with ductal carcinoma in situ, ovarian cancer and an adenoma of the colon, and in 4/5 colorectal-cancer patients investigated. However, the youngest patient, diagnosed at age 23, was a non-carrier. Loss of the wild-type BRCA1 allele was observed in 3/3 breast tissues (2 breast carcinomas and 1 ductal carcinoma in situ) but in 0/6 colorectal tissues (5 carcinomas and 1 adenoma), suggesting that BRCA1 loss is not critical for colorectal tumorigenesis. To examine the possibility that an as yet unknown gene linked to BRCA1 was involved in the colorectal cancers, chromosome 17 segregation was studied with 7 polymorphic markers encompassing a 20 cM region including BRCA1. None of these markers showed complete allele sharing among all 5 colorectal-cancer patients studied. Clinical history, mutation analysis and microsatellite instability analysis excluded a role for any of the known colorectal-cancer susceptibility genes. In 4 other Dutch families carrying the same BRCA1 mutation, only 1 colorectal-cancer case was reported, of which the carrier status is unknown.