Nasal cytokine responses to natural colds in asthmatic children.

BACKGROUND: The mechanisms by which viruses induce asthma exacerbations are not well understood. OBJECTIVE: We characterized fluctuations in nasal aspirate cytokines during naturally occurring respiratory viral infections in children with asthma. METHODS: Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured using multiplex immune assay. mRNA expression for selected markers of viral infection was measured using RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score. RESULTS: The 16 patients completed a total of 37 weeks of assessment (15 'well' weeks; 22 self-assessed 'sick' weeks). Viral infections were detected in 3 of the 'well' weeks and 17 of the 'sick' weeks (10 rhinovirus, three coronavirus, two influenza A, two influenza B, two respiratory syncytial virus, one parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1ß, CCL7/MCP-3, and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5, and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I, and IRF7 mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs, and IFN-responsive genes.

trkA modulation of developing somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in trkA knockout mice.

To investigate the nerve growth factor requirement of developing oro-facial somatosensory afferents, we have studied the survival of sensory fibers subserving nociception, mechanoreception or proprioception in receptor tyrosine kinase (trkA) knockout mice using immunohistochemistry. trkA receptor null mutant mice lack nerve fibers in tooth pulp, including sympathetic fibers, and showed only sparse innervation of the periodontal ligament. Ruffini endings were formed definitively in the periodontal ligament of the trkA knockout mice, although calcitonin gene-related peptide- and substance P-immunoreactive fibers were reduced in number or had disappeared completely. trkA gene deletion had also no obvious effect on the formation of Meissner corpuscles in the palate. In the vibrissal follicle, however, some mechanoreceptive afferents were sensitive for trkA gene deletion, confirming a previous report [Fundin et al. (1997) Dev. Biol. 190, 94-116]. Moreover, calretinin-positive fibers innervating longitudinal lanceolate endings were completely lost in trkA knockout mice, as were the calretinin-containing parent cells in the trigeminal ganglion.These results indicate that trkA is indispensable for developing nociceptive neurons innervating oral tissues, but not for developing mechanoreceptive neurons innervating oral tissues (Ruffini endings and Meissner corpuscles), and that calretinin-containing, trkA dependent neurons in the trigeminal ganglion normally participate in mechanoreception through longitudinal lanceolate endings of the vibrissal follicle.

Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.

The Tus protein of Escherichia coli is capable of arresting DNA replication in an orientation-dependent manner when bound to specific sequences in the bacterial chromosome called Ter sites. Arrest of DNA replication has been postulated to occur either by a barrier mechanism, where Tus acts as a physical block to replication fork progression, or through protein-protein interactions between Tus and some component of the replication fork. A previous mutational analysis of Tus suggested that the amino acids in the L1 loop might play a role in replication arrest. Site-directed mutagenesis of amino acids in the L1 loop and other amino acid residues on the "non-permissive" face of Tus was performed to identify residues that affected Tus function. One mutant, E47Q, gave results that are inconsistent with the barrier model, showing a greater affinity for the Ter site (with a t 1/2 of 348 min versus 150 min for wild-type Tus) but a reduced ability to arrest DNA replication in vivo. In addition to the site-directed mutagenesis studies, the tus genes of Salmonella, Klebsiella, and Yersinia were sequenced and the proteins expressed in E. coli to assess their ability to arrest DNA replication. The results presented here support a role for protein-protein interactions in Tus function, and suggest that residues E47 and E49 participate in replication fork arrest.

Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium.

Estrogen action is dependent upon the presence of specific ligand-activated receptors in target tissues. The aim of the present experiments was to compare the spatial and temporal pattern of expression of estrogen receptor beta (ERbeta) with that of ERalpha in full thickness endometrial samples (from the superficial to the basal zone) obtained from both women and rhesus macaques. Immunohistochemical localization with specific antibodies revealed that ERalpha and ERbeta were both expressed in nuclei of the glands and stroma. Consistent with previous studies, expression of ERalpha declined in the glands and stroma of the functionalis during the secretory phase. The luminal epithelium also displayed positive immunoreactivity for ERbeta. Expression of ERbeta declined in glandular cell nuclei, but not stroma, within the functionalis during the late secretory phase. Levels of expression of ERalpha and ERbeta in all cellular compartments remained unchanged in the basalis. Both receptor subtypes were detected on Western blots using proteins extracted from uterine samples obtained throughout the menstrual cycle. There was a striking contrast between the pattern of expression of ERalpha and ERbeta in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERbeta was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We conclude that estrogen action(s) within the vascular endothelium in the endometrium may be mediated via direct binding to the ERbeta isoform and that these cells could therefore be a target for agonists or antagonists that selectively target the beta form of the ER.

Afferent ingrowth and onset of activity in the rat trigeminal nucleus.

A novel in vitro preparation, consisting of the rat brainstem with the trigeminal ganglion attached, has been used to study the anatomical and functional development of the trigeminal nucleus from embryonic day (E)13 to postnatal day (P)6. Neurobiotin injections into the trigeminal ganglion showed that primary afferents had reached the trigeminal tract by E13 and had grown simple, mainly unbranched, collaterals into all levels of the nucleus by E15. By E17, these collaterals were extensively branched, with occasional boutons present. Patches of intense neurobiotin-labelled terminals, corresponding to whisker-related patterns, were first seen at E20 and became clearer over the next few days. Terminal arbours at this stage were relatively localized and densely branched, with many boutons. Responses from the trigeminal nucleus were recorded with suction electrodes, following stimulation of the trigeminal ganglion. Recordings from the main sensory nucleus showed a postsynaptic response was first present at E15. At E16, bath application of AP5 and DNQX showed that the response contained both NMDA and AMPA components, with NMDA predominating (75%). The NMDA : AMPA ratio remained high until P1, then gradually declined to 50% by P6. The postsynaptic response was also reduced by bath application of bicuculline, indicating the presence of a GABAA-mediated excitatory component. GABAergic excitation was present at all ages but was maximal from E20 to P1, the age at which whisker-related patterns are developing. It is hypothesized that both GABAergic excitation and NMDA receptor activation play a role in the consolidation of trigeminal connections, and are thus important in the development of whisker-related patterns.

Proprioceptive afferents survive in the masseter muscle of trkC knockout mice.

Peripheral innervation patterns of proprioceptive afferents from dorsal root ganglia and the mesencephalic trigeminal nucleus were assessed in trkC-deficient mice using immunohistochemistry for protein gene product 9.5 and parvalbumin. In trkC knockout mice, spinal proprioceptive afferents were completely absent in the limb skeletal muscles, M. biceps femoris and M. gastrocnemius, as previously reported. In these same animals, however, proprioceptive afferents from mesencephalic trigeminal nucleus innervated masseter muscles and formed primary endings of muscle spindles. Three wild-type mice averaged 35.7 spindle profiles (range: 31-41), six heterozygotes averaged 32.3 spindles (range: 27-41), and four homozygotes averaged 32.8 spindles (range: 26-42). Parvalbumin and Nissl staining of the brain stem showed approximately 50% surviving mesencephalic trigeminal sensory neurons in trkC-deficient mice. TrkC-/- mice (n = 5) had 309.4 +/- 15.9 mesencephalic trigeminal sensory cells versus 616.5 +/- 26.3 the sensory cells in trkC+/+ mice (n = 4). These data indicate that while mesencephalic trigeminal sensory neurons are significantly reduced in number by trkC deletion, they are not completely absent. Furthermore, unlike their spinal counterparts, trigeminal proprioceptive afferents survive and give rise to stretch receptor complexes in masseter muscles of trkC knockout mice. This indicates that spinal and mesencephalic trigeminal proprioceptive afferents have different neurotrophin-supporting system during survival and differentiation. It is likely that one or more other neurotrophin receptors expressed in mesencephalic trigeminal proprioceptive neurons of trkC knockout mice compensate for the lack of normal neurotrophin-3 signaling through trkC.

Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis.

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

Effectiveness of norgestimate and ethinyl estradiol in treating moderate acne vulgaris.

BACKGROUND: An excess of androgen is believed to contribute to development of acne in some patients. Because oral contraceptives (OCs) may reduce the active androgen level, hormonal therapy with OCs has been used successfully to treat patients with acne, although this treatment has previously not been studied in placebo-controlled trials. OBJECTIVE: Our purpose was to evaluate the efficacy of a triphasic, combination OC (ORTHO TRI-CYCLEN [Ortho-McNeil Pharmaceutical, Raritan, N.J.], norgestimate/ethinyl estradiol) compared with placebo in the treatment of moderate acne vulgaris. METHODS: Two hundred fifty-seven healthy female subjects, 15 to 49 years of age with moderate acne vulgaris, were enrolled in a multicenter, randomized, double-blind, placebo-controlled clinical trial. Each month for 6 months, subjects received either 3 consecutive weeks of the OC (i.e., tablets containing a fixed dose of ethinyl estradiol [0.035 mg] and increasing doses of norgestimate [0.180 mg, 0.215 mg, 0.250 mg]) followed by 7 days of inactive drug or placebo (color-matched tablets). Efficacy was assessed by facial acne lesion counts, an investigator's global assessment, a subject's self-assessment, and an analysis of within-cycle variation (cycle 6) in lesion counts. RESULTS: Of the 160 subjects in whom efficacy could be evaluated, the OC group showed a statistically significantly greater improvement than the placebo group for all primary efficacy measures. The mean decrease in inflammatory lesion count from baseline to cycle 6 was 11.8 (62.0%) versus 7.6 (38.6%) (p = 0.0001), and the mean decrease in total lesion count was 29.1 (53.1%) versus 14.1 (26.8%) (p = 0.0001) in the OC and placebo groups, respectively. In the investigator's global assessment, 93.7% of the active treatment group versus 65.4% of the placebo group were rated as improved at the end of the study (p < 0.001). Six of the seven secondary efficacy measures (total comedones, open comedones, closed comedones, papules, pustules, and the subject's self-assessment of study treatment) were also significantly more favorable in the OC group compared with the placebo group. CONCLUSION: An OC containing 0.035 mg of ethinyl estradiol combined with the triphasic regimen of norgestimate is a safe and effective treatment of moderate acne vulgaris in women with no known contraindication to OC therapy.

PIP: To evaluate the efficacy of a triphasic combined oral contraceptive (OC) in the treatment of moderate acne vulgaris, 231 healthy US volunteers 15-49 years of age with this dermatologic condition were enrolled in a phase III, multicenter, double-blind, placebo-controlled clinical trial. Each month for 6 months, subjects (n = 110) received either 3 consecutive weeks of Ortho Tri-Cyclen (containing a fixed dose of 0.035 mg of ethinyl estradiol and 0.180, 0.215, and 0.250 mg of norgestimate) followed by 7 days of inactive drug or placebo. The OC group showed significantly greater improvement than controls on all efficacy measures. The mean decrease in inflammatory lesion count from baseline to the sixth cycle was 11.8 (62.0%) among cases and 7.6 (38.6%) in controls, while the mean decrease in total lesion count was 29.1 (53.1%) versus 14.1 (26.8%) in the OC and placebo groups, respectively. In the investigator's global assessment, 93.7% of women in the treatment group and 65.4% of controls were rated as improved at the end of the study. Similarly, more cases than controls considered their acne "improved" at the study's end and expressed a preference for this therapy over other forms of acne treatment. These findings indicate that treatment of moderate acne vulgaris with a low-dose triphasic OC is safe and effective in women with no contraindications to OC use.

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli.

Two proteins that bind penicillin were observed in Escherichia coli infected with lambda phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C beta-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C beta-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C beta-lactamases, AmpH showed no beta-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the beta-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.

Penicillin-binding proteins and induction of AmpC beta-lactamase.

In competition assays for radiolabeled penicillin, penicillin-binding proteins (PBPs) 4, 7a, and 7b showed very high affinities for strong inducers of AmpC beta-lactamase. Loss of PBP 4 resulted in diminished inducibility. This suggests that if PBPs are involved in induction of AmpC beta-lactamase, there is probably a redundancy in function among the different PBPs.

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.

Effects of methylphenidate (Ritalin) on mammalian myocardial ultrastructure.

Previous observations have indicated lamellated ultrastructural lesions in the myocardium of a patient treated with methylphenidate (Ritalin) hydrochloride (MPH). A causal relationship between MPH exposure and these membranous changes was tested in the myocardium of rats and mice. Following injection of varying doses of MPH for different periods, myocardial ultrastructure was examined and lesions were quantified by stereological techniques. Myocardial tissue also was stained using techniques selective for acid phosphatase and for sarcoplasmic reticulum to identify possible pathogenetic mechanisms. MPH induced membrane accumulations and lamellations which were not membrane-bound and did not react for acid phosphatase, but stained positively for sarcoplasmic reticulum. Both lesions were highly focal, surrounded by normal appearing myocardial tissue. Lamellations were evident at the earliest timepoints examined and appeared to occur without lysosomal involvement. Lesions were still apparent 12 weeks after terminating MPH. These data suggest that MPH may have persistent, cumulative effects on the myocardium.

Fetal NGF augmentation preserves excess trigeminal ganglion cells and interrupts whisker-related pattern formation.

In the developing nervous system, precisely patterned connections result from mechanisms that remodel initially diffuse connections. For example, ocular dominance column formation depends upon activity-based competitive interactions. In the developing trigeminal (V) somatosensory system, injury to afferent inputs prevents somatotopic pattern formation; however, afferent impulse blockade does not. What establishes central V patterns remains unclear. As a first step in assessing the role of neurotrophins in naturally occurring death of V ganglion cells and whisker-related pattern formation, the consequences of prenatal NGF injections were evaluated. Fetal rats given NGF on both embryonic day (E) 15 and E18 had 36% more V ganglion cells than normal and lacked whisker-related patterns in the V brainstem complex at birth and through postnatal day 3, as determined by cytochrome oxidase histochemistry. Rats injected with NGF on E16 or on E18, or with vehicle had normal ganglion cell numbers and brainstem patterns. Animals injected with antibodies to NGF or an NGF receptor had reduced ganglion cell numbers and normal brainstem patterns. These findings suggest that naturally occurring cell death in the V ganglion is neurotrophically regulated and that this process impacts upon somatotopic pattern formation in the V brainstem complex. Results of anterograde tracing experiments in NGF-augmented animals suggest that pattern disruptions are due to an absence of whisker-related patterning in the central projections of V ganglion cells. Moreover, single primary afferent collaterals labeled by Neurobiotin injections in the V ganglion did not have widespread or unusually complex arbors. Thus, NGF may affect V pattern formation by preserving or inducing projections to brainstem regions that normally come to lack such projections, such as the spaces normally demarcating neighboring whisker primary afferent projections.

Artifactual processing of penicillin-binding proteins 7 and 1b by the OmpT protease of Escherichia coli.

Penicillin-binding proteins (PBPs) were visualized in strains of Escherichia coli that carried mutations in one or more of the following protease genes: tsp, degP, ptr, and ompT. In the absence of a functional ompT gene, PBPs 1b alpha and 7 were not processed to the shortened forms 1b beta and 8, respectively. Cleavage of PBPs 1b alpha and 7 could be restored by introduction of a plasmid carrying the wild-type ompT gene. These PBPs were processed only after cell lysis or after membrane perturbation of whole cells by freeze-thaw, suggesting that the cleavage was a nonspecific artifact due to contact with OmpT, an outer membrane protease, and that such processing was not biologically significant in vivo. The degradation of other PBPs during purification or storage may also be effected by OmpT.

NGF augmentation rescues trigeminal ganglion and principalis neurons, but not brainstem or cortical whisker patterns, after infraorbital nerve injury at birth.

Prior studies indicate that neonatal nerve injury kills many trigeminal (V) first- and second-order cells, and interrupts pattern formation in the brainstem and cerebral cortex. Yet it is not known whether effects upon cell survival and pattern formation are causally related. To determine whether axotomized V ganglion cells can be rescued by an exogenous trophic agent, rats received 5 mg/kg of nerve growth factor (NGF) prior to, and every day after, infraorbital nerve section on the day of birth until sacrifice on postnatal day (PND) 1, 3, 5, 7, or 14. Other animals received identical lesions without NGF. Ganglion cell numbers were significantly reduced by PND1 in pups not given NGF, while NGF-treated rats displayed no significant cell loss through PND7. However, NGF did not permanently rescue V neurons because ganglion cell numbers were reliably reduced by PND14. Cell numbers in V nucleus principalis were reduced by PND1 in pups not given NGF, while NGF-treated animals displayed no cell loss through PND14. NGF's rescue of second-order cells is probably an indirect effect of NGF action upon V ganglion cells because, in other newborns, NGF failed to maintain principalis cells after direct lesion of the left V ganglion. To determine whether preventing cell death permits whisker-related pattern formation, other rats also received NGF prior to and after infraorbital nerve section at birth. After 3-14 days, patterns were assessed in the brainstem and cortex with cytochrome oxidase histochemistry and serotonin immunocytochemistry. Whisker-related patterns failed to develop as in cases not given NGF. These data indicate that communication with the periphery is necessary for the maintenance of central whisker-related patterns. They also suggest that V ganglion cells can be rescued, albeit temporarily, from rapid injury-induced death by NGF, thereby delaying injury-induced cell death in nucleus principalis. However, the mechanism(s) responsible for injury-induced pattern alterations in the developing V system remains to be elucidated.

Structure-function relationships in rat brainstem subnucleus interpolaris. X. Mechanisms underlying enlarged spared whisker projections after infraorbital nerve injury at birth.

Prior studies indicate that the central projections of noninfraorbital vibrissae occupy greater than normal transverse areas in the rat trigeminal brainstem complex after infraorbital nerve section at birth. Here, we assessed the development of this phenomenon and possible underlying mechanisms. Cytochrome oxidase patches representing spared supraorbital (SO) or posteroorbital (PO) whiskers in the trigeminal subnucleus interpolaris (SpVi) were not reliably larger than those on the control side 24 hr after the infraorbital lesion. By 72 hr, SO and PO patches were 91% and 28% larger than those on the control side. Reliable increases were also observed on postnatal day 5 (PND5), PND7, and PND10 for the SO (59%, 65%, 66%) and PO (23%, 44%, 51%) patches. To test the hypothesis that central reorganization reflects the maintenance of peripheral supernumerary axons, myelinated and unmyelinated axons in SO vibrissa follicles were counted at PND0, PND7, PND17, and PND60. A corollary hypothesis, that peripheral regeneration errors result in both SO and surviving infraorbital axons, contributing to central SO patches, was tested with retrograde double-labeling methods. Both hypotheses were rejected. Thus, enlargement of SO patches is not due to either the maintenance of an immature peripheral innervation pattern, or regeneration of infraorbital axons into SO follicles. To determine if the enlargement of SO and PO patches produced by infraorbital nerve section is due to an activity-dependent competitive disadvantage imposed upon infraorbital afferents, TTX or bupivicaine was applied to the intact infraorbital nerve over the first 5-9 postnatal days. Brainstem maps developed normally and SO and PO patch areas were unaffected. Thus, impulse activity-based mechanisms do not appear to contribute to injury-induced patch enlargement. To test the hypothesis that patch enlargement is due to central collateral reorganization, intra-axonal recording and staining methods were applied to control and spared-whisker primary afferents in adult rats. Total bouton or collateral numbers did not differ in SpVi; however, arbor areas were reliably larger in experimental (14,879 +/- 350 microns 2) versus control (5527 +/- 1811 microns 2) fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

2-DG uptake patterns related to single vibrissae during exploratory behaviors in the hamster trigeminal system.

Stimulation of one or several whiskers activates discrete foci throughout the trigeminal (V) neuraxis. These foci contribute to patterns, corresponding to the patterns of vibrissae, that have been directly related to aggregates of cells and axon terminals in the "barrel" cortex. Here, we combine high-resolution, 2-deoxyglucose (2DG) mapping and cytochrome oxidase (CO) staining to determine whether the known pattern of V primary afferent projections is sufficient to deduce the functional activation of their targets during exploratory behavior. Four adult hamsters had all of their large mystacial vibrissae trimmed acutely, except for C3 on the left, and B2 and D4 on the right; in two others, the left C3 and right A1 and E4 whiskers were spared. After fasting overnight, 2DG was injected and the animals behaved freely in the dark for 45 minutes. The brainstem, thalamus, and cortices were sectioned, then processed for both CO staining and 2DG autoradiography. Image-processing microscopy was used to separate the autoradiographic silver grains from the histochemical staining. CO patches were patterned in a whisker-like fashion in the full rostrocaudal extent of V nucleus principalis and in caudal portions of spinal V subnuclei interpolaris and caudalis, but absent in subnucleus oralis. 2DG silver grains were densest above those CO patches in the pattern corresponding to the active whiskers. There were no consistent 2DG foci in subnuclei oralis or rostral caudalis. In these same cases, prominent 2DG labeling was restricted to the appropriate barrels in the contralateral cortex. Only one case, however, displayed a clear and appropriate region of heightened 2DG uptake in contralateral ventroposteromedial thalamus (VPM) and the adjacent part of the reticular thalamic nucleus. Patterns of increased glucose utilization with single whisker stimulation are well matched to the CO patterns that mirror distributions of neurons associated with a vibrissa in the V brainstem complex, thalamus, and cortex. Single whiskers are represented by relatively homogeneous longitudinal columns of 2DG labeling in the V brainstem nuclei. The columns are not continuous through the axial extent of the V brainstem complex; rather, they occur separately within principalis, interpolaris, and caudalis. While whisker columns were consistently labeled in interpolaris and caudalis in all animals, the labeling was increasingly variable in principalis, barrel cortex, and VPM, respectively. This suggests that the behaving animal can and does significantly modulate activity in this major, synaptically secure pathway.

Infraorbital nerve blockade from birth does not disrupt central trigeminal pattern formation in the rat.

We tested the hypothesis that patterned primary afferent impulse activity during early postnatal periods is necessary for central trigeminal pattern formation. Newborn rats had their whiskers trimmed daily and new slices of slow release polymer containing the sodium channel blocker, tetrodotoxin, were placed under the infraorbital nerve every 8 h for up to 9 days. Electrophysiological recordings indicated that trigeminal ganglion cells were unresponsive to peripheral stimuli and chronically silenced. Trigeminal ganglion cell numbers were unaffected by nerve blockade. Cytochrome oxidase staining patterns in the trigeminal brainstem complex, thalamus, and barrel cortex were normal on postnatal day 1, 3, 5, 7, or 9 (n = 4 each). Whisker-related patches were of normal sizes and staining densities. Similar negative results were obtained in 9 rats in which whiskers were trimmed daily and the long-acting local anesthetic bupivacaine was injected into the whisker pad at 2.5- to 4-h intervals from birth to sacrifice on postnatal day 5-9. Cytochrome oxidase staining patterns and patch properties again did not differ from normal. Thus, trigeminal pattern formation occurs even when the entire infraorbital nerve is silenced from birth.