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Influenza virus was shown to disseminate from the respiratory tract of nasally infected ferrets to a surgically formed, subcutaneous tracheal pouch. Conversely, ferrets infected via the pouch shed virus from the upper respiratory tract. These events occurred within the first 24 h after infection. Passively administered ferret antibody to virus did not prevent the dissemination.
There is little in the literature to support recommendations for the use of steroids and vasoconstrictors in the treatment of postintubation croup. We developed a ferret model to study this condition. Ninety-eight ferrets were used in these experiments, which were undertaken to evaluate the efficacy of steroids and/or a long-acting vasoconstrictor, to develop dose-response curves for dexamethasone sodium phosphate if it proved efficacious, and to evaluate the possible synergistic effects of combination therapy. With this animal model, we were able to document a statistically significant early effect of dexamethasone sodium phosphate, but only when administered in large doses (greater than 0.5 mg/kg). Our data also support the tapering of these large doses within a 20-hour period. The long-acting vasoconstrictor oxymetazoline hydrochloride has a clear and statistically significant beneficial effect that is apparent by 20 hours and continues through 40 hours. We did not find any additive or synergistic effect of the combination of dexamethasone and oxymetazoline. Further studies are underway in our laboratory to elucidate other aspects of this interesting syndrome.
Immunohistochemical methods were used to study the distribution and time-course of appearance of cells expressing glutamate and alpha-amino-3-hydroxy-5-methyl-4-isoaxazole propionic acid (AMPA)-type glutamate receptors (GluR1 and GluR2/3) during development of the ferret visual cortex. Glutamate is present in many neurons in the ventricular zone, intermediate zone, developing cortical plate, and marginal zone as early as embryonic day (E) 34 (birth is at E41 in ferrets). Glutamate attains its adult distribution coincident with the completion of cellular migration. By contrast, GluR1 immunoreactivity emerges more slowly. By birth, GluR1 immunoreactivity is present only in a few neurons in the marginal zone and ventricular zone but is abundant in the marginal zone and subplate, where synaptogenesis commences. The number and staining intensity of GluR1-positive cells increases dramatically during the first two postnatal weeks and is maximal between the second and third week, before slowly declining to adult levels. Cortical cells immunopositive for GluR2/3 follow a similar pattern, although their distribution differs: GluR2/3-positive cells are mainly pyramidal cells. During the first postnatal week, GluR2/3 is also transiently present in fibers in the intermediate zone, which at this stage contains many thalamocortical and callosal and corticofugal axons. The abundance of glutamate at fetal stages, especially in the ventricular zone, is consistent with the previously proposed role of glutamate in mediating trophic effects in vivo, as previously demonstrated in vitro. The expression of AMPA receptors, as well as their transient overexpression, confirms the results of in situ hybridization studies and may imply a developmental role in neuronal differentiation for these receptors, in addition to their mature role in mediating cortical transmission.
The goal of this study was to describe the development of gamma-aminobutyric acid (GABA)-containing neurons in visual and auditory cortex of ferrets. The laminar and tangential distribution of neurons containing excitatory, inhibitory, and neuromodulatory substances constrain the potential circuits which can form during development. Ferrets are born at an early stage of brain development, allowing examination of inhibitory circuit formation in cerebral cortex prior to thalamocortical ingrowth and cortical plate differentiation. Immunocytochemically labelled nonpyramidal GABA neurons were present from postnatal day 1 throughout development, in all cortical layers, and generally followed the inside-out pattern of neuronal migration into the cortical plate. Prior to postnatal day 14, pyramidal neurons with transient GABA immunoreactivity were also observed. The density of Nissl-stained and GABA-immunoreactive neurons was high early in development, declined markedly by postnatal day 20, then remained relatively constant until adulthood. However, examination of the proportion of GABA neurons revealed an unexpected late peak at postnatal day 60, then a decrease in adulthood. Visual and auditory cortex were similar in most respects, but the peak at postnatal day 60 and the final proportion of GABA neurons was higher in auditory cortex. The late peak suggests that inhibitory circuitry is stabilized relatively late in sensory cortical development, and thus that GABA neurons could provide an important substrate for experience-dependent plasticity at late stages of development.
Chemical characteristics of developing neurons in the superior olivary complex of the ferret were analyzed using immunohistochemical methods. The present report of calcium-binding proteins in the developing and adult superior olivary complex shows distinct distribution patterns for parvalbumin, calbindin, and calretinin in the lateral superior olivary nucleus (LSO) of the developing ferret that correspond to distribution patterns for different projection cell types and neurotransmitters. In the neonate, there was an initial complementary distribution of calcium-binding proteins between the shell and core of the body of the developing LSO. Parvalbumin and calbindin-immunoreactive cells were present in the shell, whereas calretinin-immunoreactive cells were restricted to the core of the LSO. Gamma amino butyric acid (GABA), but not glycine, immunoreactive cells were distributed similarly in the shell of the LSO in the neonate. There were, in addition, reciprocal medial-to-lateral gradients of parvalbumin and calbindin-immunoreactive cells in the LSO shell of the neonate. These complementary patterns in the LSO were transient, however, and by the end of the second postnatal week, each calcium-binding protein differed markedly in its cellular distribution in the superior olive, including the LSO. GABA-immunoreactive cells also were restricted transiently to the shell of the LSO in neonates. The radial segregation of transient calcium-binding expression in LSO cells was orthogonal to the medial-to-lateral axis in the LSO and, therefore, parallels fibrodendritic layers and presumed isofrequency planes of the LSO. The early postnatal segregation of calcium-binding proteins in the isofrequency axis was congruent with the gradients of contralateral and ipsilateral projection cell types in adult LSO. It seems likely that developmental mechanisms regulate expression of calcium-binding protein and neurotransmitter phenotypes and that these mechanisms operate in development within the isofrequency axis as well as along the tonotopic axis of this auditory nucleus.
Airway mucus hypersecretion is in part a response to infection and inflammation. Pseudomonas aeruginosa infection is nearly universal in advanced cystic fibrosis (CF) lung disease. Mucoid strains of P. aeruginosa produce an exopolysaccharide product called alginate. The purpose of this study was to determine whether P. aeruginosa alginate stimulates secretion from mucous or serous cells in the ferret trachea exposed to alginate at concentrations reported to be present in the CF airway. We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin secretion and spectrophotometry to measure lysozyme secretion from isolated ferret tracheal segments. Purified Pseudomonas aeruginosa alginate stimulated mucin and lysozyme secretion in a dose-dependent fashion (mucin = +111%: P = 0.003; lysozyme = +20%: P = 0.024 at 200 microg/mL). This stimulated secretion was not due to proteolytic activity, and alginate exposure did not produce ultrastructural damage to the trachea. We conclude that alginate may contribute to mucus hypersecretion and respiratory morbidity associated with P. aeruginosa infection in patients with CF.
Ferret parotid saliva has been analysed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) to determine, for the first time, its protein composition. SDS-PAGE, in combination with Coomassie Brilliant Blue (CBB) staining, revealed up to 20 bands and the patterns were characterised by major protein constituents of Mr 105000, 51000, 47000, 33000, 22000 and 16 400 common to all samples from all animals. Sequential samples collected from the same animal during prolonged stimulation of the parasympathetic nerve (40 min at 40 Hz) showed subtle but reproducible protein changes. Saliva collected from different animals varied widely in the amount of a protein Mr 66000. 2-DE, in combination with silver staining, revealed up to 300 spots and the patterns were characterised by major protein constituents of Mr 105000 (pI 6.3-7.2), Mr 66000 (pI 4.7-5.3), Mr 51000 (pI 5.0-5.7), Mr 47000 (pI 6.0-7.5), and Mr 33000 (pI 4.7-6.0). Many of the polypeptide spot clusters consisted of one or more horizontal strings of spots suggesting extensive microheterogeneity. Both SDS-PAGE and 2-DE indicated that the protein patterns of ferret parotid saliva evoked by electrical stimulation of the parasympathetic nerve in the absence or presence of atropine are similar, i.e., the protein composition of the atropine-resistant nonadrenergic, noncholinergic (NANC) secretion is similar to that of saliva evoked in the absence of muscarinic receptor blockade.
The motor control of the lower esophageal sphincter (LES) is critical for normal swallowing and emesis, as well as for the prevention of gastroesophageal reflux. However, there are surprisingly few data on the central organization and neurochemistry of LES-projecting preganglionic neurons. There are no such data in ferrets, which are increasingly being used to study LES relaxation. Therefore, we determined the location of preganglionic neurons innervating the ferret LES, with special attention to their relationship with gastric fundus-projecting neurons. The neurochemistry of LES-projecting neurons was also investigated using two markers of "nontraditional" neurotransmitters in vagal preganglionic neurons, nitric oxide synthase (NOS), and dopamine (tyrosine hydroxylase: TH). Injection of cholera toxin B subunit (CTB)-horseradish peroxidase (HRP) into the muscular wall of the LES-labeled profiles throughout the rostrocaudal extent of the dorsal motor nucleus of the vagus (DMN) The relative numbers of profiles in three regions of the DMN from caudal to rostral are, 43 +/- 5, 67 +/- 11, and 113 +/- 30). A similar rostrocaudal distribution occurred after injection into the gastric fundus. When CTB conjugated with different fluorescent tags was injected into the LES and fundus both labels were noted in 56 +/- 3% of LES-labeled profiles overall. This finding suggests an extensive coinnervation of both regions by vagal motor neurons. There were significantly fewer LES-labeled profiles that innervated the antrum (16 +/- 9%). In the rostral DMN, 15 +/- 4% of LES-projecting neurons also contained NADPH-diaphorase activity; however, TH immunoreactivity was never identified in LES-projecting neurons. This finding suggests that NO, but not catecholamine (probably dopamine), is synthesized by a population of LES-projecting neurons. We conclude that there are striking similarities between LES- and fundic-projecting preganglionic neurons in terms of their organization in the DMN, presence of NOS activity and absence of TH immunoreactivity. Coinnervation of the LES and gastric fundus is logical, because the LES has similar functions to the fundus, which relaxes to accommodate food during ingestion and preceding emesis, but has quite different functions from the antrum, which provides mixing and propulsion of contents for gastric emptying. The presence of NOS in some LES-projecting neurons may contribute to LES relaxation, as it does in the case of fundic relaxation. The neurologic linkage of vagal fundic and LES relaxation may have clinical relevance, because it helps explain why motor disorders of the LES and fundus frequently occur together.
Nitric oxide (NO) is a diffusible neurotransmitter that has been implicated in key developmental events, including the refinement of retinogeniculate axons into ON/OFF sublayers in the ferret lateral geniculate nucleus (LGN), and in the formation of eye-specific laminae in other species. To understand the role of NO in the LGN, it is critical to fully characterize the pattern of brain nitric oxide synthase (bNOS) expression within the nucleus, including the phenotype of the neural elements that express it. We have examined the temporal and spatial pattern of bNOS expression in the ferret LGN during the first 6 weeks of postnatal development, and in the adult, by detecting bNOS with a monoclonal antibody as well as beta-nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. We have found that bNOS is expressed in neurons in the A laminae of the LGN as early as postnatal day 7 (P7), a time coincident with eye-specific segregation of retinal axons. This expression continues through P35, with peak somatodendritic expression at P21. Fluorescent double labeling using antibodies to bNOS and glutamic acid decarboxylase indicate that bNOS is expressed in gamma-aminobutyric acid-ergic interneurons within the A laminae. Electron microscopic examination of bNOS-labeled cells showed synaptic contacts from terminals with two distinct morphologic profiles. Expression of bNOS within interneurons that receive contacts from multiple sources indicates that the synaptic circuitry associated with bNOS activation and the potential targets of NO may be more complex than originally thought and supports a potential new role for interneurons as cellular intermediaries in the refinement of pathways in the LGN. Our findings broaden the window of time that bNOS may be active within the developing LGN, suggesting an expanded role for NO during early postnatal development.
In this study, we used image analysis to assess changes in calretinin immunoreactivity in the lateral (LSO) and medial (MSO) superior olivary nuclei in ferrets 2 months after unilateral cochlear ablations at 30-40 days of age, soon after hearing onset. These two nuclei are the first significant sites of binaural convergence in the ascending auditory system, and both receive direct projections from the deafferented cochlear nucleus. Cochlear ablation results in a decrease in the overall level of calretinin immunostaining within the LSO ipsilaterally compared with the contralateral side and with control animals and within the MSO bilaterally compared with control ferrets. In addition, the level of calretinin immunostaining ipsilaterally within neurons in the LSO was significantly less in cochlear ablated than control animals. In contrast, there was no effect of cochlear ablation on the level of calretinin immunostaining within neurons either in the contralateral LSO or in the MSO. These results are consistent with a downregulation in calretinin within the neuropil of MSO bilaterally and LSO ipsilaterally, as well as a downregulation in calretinin within somata in the ipsilateral LSO as a result of unilateral cochlear ablation soon after hearing onset. Thus, cochlear-driven activity appears to affect calcium binding protein levels in both neuropil and neurons within the superior olivary complex.
Thalamocortical axons segregate into ocular dominance columns several weeks before the onset of critical period plasticity in ferret visual cortex, a stage characterized by anatomical changes in thalamic input as a consequence of abnormal visual stimulation. In search of possible anatomical correlates of this plasticity, we examined, at electron microscope resolution, the morphology and the synapsing and target selection properties of thalamic axons in ferret visual cortex during and after the critical period. Adult thalamocortical terminals visualized by anterograde tract-tracing display significantly larger cross-section areas than terminals at postnatal day (P) 35, P40, and P49 critical period ages. They are also significantly larger than nonthalamocortical terminals, which attain an adult-like size distribution by P40. The synaptic zones of adult thalamocortical terminals are significantly larger than those of critical period terminals. Perforated and invaginated synapses are encountered frequently on thalamic axons in both adulthood and the P40-49 age group. This result contradicts the view that synaptic perforations and spinules are indicative of a capacity for plasticity. It also suggests that at least some morphological features of thalamic terminals attain maturity on a developmental schedule that is independent of critical period plasticity. Connectivity properties of labeled axons, however, suggest an active role for thalamocortical axons in the critical period. In P40, P49, and adult brains, 23%, 17%, and 9%, respectively, of all thalamocortical synapses contact GABAergic interneurons, suggesting that thalamic input is more strongly involved in driving inhibitory circuits in young ages. Furthermore, thalamocortical axons in P35-49 brains form about 60% more synapses per axon length than in adult brains, suggesting that stabilization of thalamic synapses at the end of the critical period may be accompanied by a reduction of synaptic contacts, as well as a reorganization of postsynaptic circuit selectivity.
Interareal feedback connections are a fundamental aspect of cortical architecture, yet many aspects of their organization and functional relevance remain poorly understood. Previous studies have investigated the topography of feedback projections from extrastriate cortex to macaque area 17. We have extended this analysis to the ferret. We made restricted injections of cholera toxin B (CTb) into ferret area 17 and mapped the distribution of retrogradely labeled cells in extrastriate cortex. In addition to extensive label spreading within area 17, we found dense cell label in areas 18, 19, and 21 and the suprasylvian cortex and sparser connections from the lateral temporal and posterior parietal cortex. We made extensive physiological assessments of magnification factors in the extrastriate visual cortex and used these measures to convert the spread of labeled cortex in millimeters into a span in degrees of visual field. We also directly measured the visuotopic extents of receptive fields in the regions containing labeled cells in cases in which we made both CTb injections and physiological recordings in the same animals; we then compared the aggregate receptive field (ARF) of the labeled region in each extrastriate area with that of the injection site. In areas 18, 19, and 21, receptive fields of cells in regions containing labeled neurons overlapped those at the injection site but spanned a greater distance in visual space than the ARF of the injection site. The broad visuotopic extent of feedback connections is consistent with the suggestion that they contribute to response modulation by stimuli beyond the classical receptive field.
The anterograde transport of 3H proline and of horseradish peroxidase has been used to study the retinogeniculate pathway in normal adult ferrets and in young ferrets during postnatal development. the lateral geniculate nucleus in adults shows a characteristic "carnivore" pattern, with layers A, A1, C, C1, C2, and C3, and a medial interlaminar nucleus recognizable either cytoarchitectonically or on the basis ofth retinogeniculate innervation. In addition, there is a well-defined, rather large perigeniculate nucleus. At birth the lateral geniculate nucleus is unlaminated and essentially all parts are reached by afferents from both eyes. The crossed component is by far the larger. It extends from the optic tract medially well into the perigeniculate field, in contrast to the uncrossed component which barely reaches the perigeniculate field. During the first 3 postnatal days the uncrossed fibers restrict their arbors to a small posterior and medial region, the precursor of the biocular segment of the nucleus. The crossed fibers gradually retreat from the region within which the uncrossed fibers have concentrated. Between the fourth and eighth postnatal days the field occupied by the ipsilateral component expands again to form a major focus that will define lamina A1 and a minor focus that will define C1. At this stage the crossed and the uncrossed fibers overlap at the borders of lamina A1 and the whole region of lamina C1 is also occupied by arbors of the crossed component. The perigeniculate field becomes clearly distinguishable from the lateral geniculate nucleus and the medial interlaminar nucleus is becoming clearly recognizable between days 3 and 8. Between days 8 and 15 the cytoarchitectonic borders between layers A and A1 become clearly defined, but the retinogeniculate axons from each eye still extend across this border. These axons retreat into their appropriate lamina after the 15th postnatal day an the nucleus reaches its essentially adult structure by about the fourth postnatal week. Segregation of retinofugal axons in the C layers occurs after segregation in the A layers, but many of the cells within the C layers show signs of cytological maturity earlier than those of the A layers. The nucleus undergoes a series of migrations and changes of shape as the ipsilateral and contralateral components become segregated. Whereas in teh newborn the nucleus is roughly comma-shaped and on the lateral aspect of the dorsal thalamus, in the adult it is "L"-shaped and mainly on the posterior aspect of the dorsal thalamus.
The projection of the visual field onto the dorsal lateral geniculate nucleus (LGN) of the ferret was mapped electrophysiologically. The nucleus contains a single orderly map of the contralateral visual hemifield. The upper visual field is represented dorsally and rostrally in the nucleus; central fields are found in the medial and caudal sections of the LGN; and peripheral fields are represented most laterally. The ipsilateral eye is represented in laminae A1 and C1 up to eccentricities of 20-30 degrees. Lines of projection run perpendicular to the laminar borders. The ferret LGN resembles that of the cat rotated approximately 110 degrees clockwise in the sagittal plane, viewing the right nucleus from its lateral aspect; it differs from the cat in having a larger monocular segment.
The present report describes the intrinsic and extrinsic cortical connectivity of striate cortex (area 17) in the ferret. Injections of horseradish peroxidase demonstrate periodic intrinsic connections over an extent of 2.5-3.0 mm, mainly in the supragranular layers but also occurring secondarily in layer 5. These connections have a stripelike configuration, with a center-to-center spacing of 0.5-0.7 mm. Their laminar distribution and stripelike configuration resemble the pattern in the cat (Gilbert and Wiesel, '83), another member of the carnivore family, but not that in monkeys. In both macaque and squirrel monkeys, these connections have a bilaminar distribution in layers 2-3 and 4B, and a more complicated latticelike geometry (Rockland and Lund, '83). Their interperiod spacing, of about 0.5 mm, however, is relatively constant across species. Extrinsic connections in the ferret link striate cortex with territories probably homologous to feline areas 18 and 19, and to the suprasylvian region. Callosal connections extend on the lateral surface about 1.5 mm into area 17 and 4.0 mm into area 18 beyond their common border. There are homotopical connections between striate cortices and heterotopical connections from at least areas 18 and 19 to contralateral area 17. In addition to gray matter connections, intracortical injections also result in labeled interstitial neurons in the subgriseal white matter. These occur both subjacent to an injection site in area 17, and below labeled foci in area 18 projecting back to area 17, as if interstitial neurons shared the connectivity of overlying layer 6.
The organization of the auditory brainstem in adult, darkly pigmented ferrets was studied by using the retrograde transport of the lectin wheat germ agglutinin-horseradish peroxidase injected into one inferior colliculus. Retrogradely labelled neurons were found bilaterally in every nucleus of the auditory brainstem. The greatest number of labelled neurons was found in the cochlear nuclei contralateral to the injection site, the ipsilateral medial superior olivary nucleus, both lateral superior olivary nuclei, the ipsilateral ventral nucleus of the lateral lemniscus, both dorsal nuclei of the lateral lemniscus, and the contralateral inferior colliculus. Quantitative assessment of the projections from the cochlear nuclei showed that the number of contralaterally projecting neurons exceeded the number of ipsilaterally projecting neurons by about 50 to one. This ratio remained relatively stable over a wide range of volumes of injected lectin, whereas the absolute number of labelled neurons on each side varied by at least twofold for a constant volume of lectin. These results provide basic data on the ferret auditory system and demonstrate quantitatively some properties of the projections between the cochlear nucleus and the inferior colliculus.
Anatomical and electrophysiological mapping techniques were used to determine topographic organization and arrangement of ocular dominance columns in the primary visual cortex of ferrets. From its border with area 18 on the posterior lateral gyrus, area 17 extends around the caudal pole of the hemisphere and over the splenial gyrus to the caudal bank of the splenial sulcus. The visuotopic map is oriented with the isoazimuth lines approximately parallel to the long axis of the posterior lateral gyrus and the isoelevation lines approximately perpendicular to the isoazimuths. Central azimuths are represented on the posterior lateral gyrus and peripheral azimuths are represented on the splenial gyrus; the inferior visual field maps medially and the superior visual field maps laterally. As in other species, the representation of the central visual field is expanded. The ferret has a considerable degree of binocular vision. Receptive fields driven through the ipsilateral eye extended more than 20 degrees into the contralateral visual field. Within the region of area 17 corresponding to the binocular portion of the visual field, tritiated proline injected into one eye transneuronally labelled an ipsilateral projection as a series of patchy bands roughly complementary to gaps in the labelled contralateral projection. Physiological ocular dominance columns were evident as well in that neurons and groups of neurons recorded in this region showed clustered ocular dominance preferences. Most single neurons studied were binocularly driven.
Dendritic morphology and development in the medial superior olivary nucleus of the ferret were studied using the Golgi method. In the adult ferret most medial superior olivary neurons had disk-shaped dendritic fields. These dendritic fields were oriented such that cells in a coronal plane of section appeared bipolar with major lateral and medial dendritic axes. In the horizontal plane the dendrites radiated about the soma. Dendrites of principal cells branched distally into tufts of numerous, tertiary processes that were beaded and thin. Peripheral cells in the fiber mantle encircling the nucleus were generally spindle-shaped or tripolar and lacked the tufted dendrites of principal cells. The dendrites of these peripheral cells coursed parallel to the nucleus both dorsoventrally and rostrocaudally. Horizontally oriented dendrites were observed even at birth for some cells in the medial superior olivary nucleus and bipolar dendritic fields were typical of most cells by the end of the second postnatal week. Dendrites of immature cells varied in caliber and radiated in all directions from the soma. Around postnatal days 8-10 transient appendages appeared on the soma and dendrites, first proximally and then more distally. These appendages persisted until the first postnatal month. Cell size and dendritic radius increased markedly during this same period. Postnatal days 28-30 were marked by the first appearance of tufts of tertiary dendritic branches. The tendril-like processes continued to increase in length until about the end of the second postnatal month.