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Development of glycinergic cells and puncta in nuclei of the superior olivary complex of the postnatal ferret.

The distribution of glycine-immunopositive cells and axonal endings was studied in the adult and early postnatal ferret superior olive. As in other species, the most prominent glycine-immunopositive cell group in the adult ferret superior olive was the medial nucleus of the trapezoid body. Other darkly immunostained cells were present, although more scattered, in most periolivary regions, including the lateral and ventral trapezoid body nuclei. In the lateral superior olivary nuclei, glycine-immunopositive cells were intermingled with immunonegative cells. A comparable population of cells in the ipsilateral lateral superior olivary nucleus was retrogradely labeled in cases with unilateral injections of tritiated glycine in the inferior colliculus. Glycine-immunopositive puncta were widely distributed in the neuropil in most periolivary regions, including dense accumulations in the dorsomedial periolivary region and ventral and lateral nuclei of the trapezoid body. In the lateral and medial superior olivary nuclei, immunopositive puncta were distributed around the principal cells in characteristic perisomatic halos. In postnatal ferrets, immunopositive cell bodies were first observed by postnatal day 7 and were distributed in regions comparable to regions in the adult, with the exception that immunopositive cells in the lateral superior olivary nucleus did not appear until about postnatal day 28. There was diffuse staining in the neuropil in principal and periolivary nuclei by postnatal day 7. During the third postnatal week, the immunostaining in the neuropil began to take on a more granular appearance and immunopositive puncta could be seen by postnatal day 35. In the lateral and medial superior olivary nuclei, the earliest distribution of immunostaining in the neuropil was nonuniform, being greater in the high-frequency, medial, and ventral regions, respectively. The density gradient in these areas was gradually eliminated over the next 2 postnatal weeks as immunostained processes and endings appeared over greater portions of the nuclei.

Animals↗

Seasonal fiber growth cycles of ferrets (Mustela putorius furo) and long-term effects of melatonin treatment.

Pelage cycles of ferrets are poorly documented, although it is clear that their timing is sensitive to daylength, mediated by pineal melatonin. Hair follicles were monitored histologically in ferrets from 3 to 19 months of age in order to describe naturally occurring changes in follicle growth status and follicle number over three successive cycles of fur growth. Melatonin was administered to some of these animals in late summer to determine the long-term effects of perturbation of hormonal control. Circulating melatonin was elevated for approximately 50 days by 8-mg continuous release implants. Treated animals grew both their first winter coat, and subsequent summer coats 18 days in advance of untreated controls, but this effect did not extend to the second winter coat. Reimplantation the following year induced an advancement of the autumn follicle growth as in the 1st year. Autumn fiber growth occurred at similar times in untreated males and females, and response to melatonin did not differ between sexes. Hair follicle regression and shedding during the natural spring molt was also contemporaneous in males and females, but fiber regrowth occurred 4-6 weeks later in males as compared with females, suggesting that reproduction-related factors affect fiber growth initiation, and that fiber growth and shedding are physiologically distinct processes. Melatonin implants in autumn also affected reproduction in the spring, advancing oestrus by 3-4 weeks. These results show that interference with photoperiodic and hormonal control mechanisms in ferrets can affect pelage and reproductive cycles for up to 10 months.

Animals↗

Nuclear transfer of M-phase ferret fibroblasts synchronized with the microtubule inhibitor demecolcine.

The development of reconstructed embryos following nuclear transfer (NT) appears to be dependent upon a variety of factors, including cell cycle synchronization between the donor nucleus and recipient oocyte. Here we use the microtubule inhibitor, demecolcine, to synchronize ferret fibroblasts in metaphase (M-phase) in order to match their cell cycle position with that of the recipient oocyte at the time of NT. The fibroblasts were obtained from 28-day fetuses and cultured for 1-30 days prior to NT. Fibroblast cultures were treated with 0.05 microg/ml of demecolcine for 3 hr or overnight (14-16 hr) after various times in culture to determine the optimal conditions for M-phase synchronization. The percentage of G2/M-phase cells in demecolcine-treated cultures was significantly greater than that found in untreated cultures (P<0.05). Optimally synchronized M-phase fibroblasts were collected by mitotic shake-off and evaluated for their effectiveness in NT. M-phase somatic cell-derived NT embryos reconstituted by electrofusion or microinjection underwent implantation and formed fetuses at similar rates (5.4% vs. 3.4%, and 1.8% vs. 1.2%, respectively); however, no NT embryos developed to term. In summary, these data demonstrate two important points. First, demecolcine treatment effectively synchronizes ferret fibroblasts in M-phase of the cell cycle; and second, these somatic cells are capable of driving embryo development following NT. Our results should facilitate the development of cloned ferrets as an animal model for human lung disease such as influenza and cystic fibrosis.

Analysis of Variance↗

The effect of trypan blue on the development of the ferret and rat.

The teratogenic effects of trypan blue given to pregnant rats and ferrets at equivalent stages of embryonic development are described. In the rat the well known teratogenicity of the dye at 8.5 days of gestation and the surprising cessation of teratogenic effect at 11.5 days (20+ somites) is confirmed. In the ferret the drug is teratogenic both at 13 and 18 days of gestation though the principal abnormalities seen are different to those described in the rat. It has been suggested that the teratogenic action of trypan blue in rats is due to an interference in the function of the yolk sac which is an organ of histiotrophic nutrition and provides the principal source of nutrition for the 8.5-day embryo that is largely replaced by a functional chorio-allantoic placenta after 11.5 days of gestation. In ferrets histiotrophic nutrition does not involve an inverted yolk sac placental system. In this species endometrial symplasma provides histiotroph which is ingested by the invading trophoblast. Histiotrophic nutrition is still of great importance at 18 days and morphological evidence indicates that chorio-allantoic placentation becomes important by about 20 days (35+ somites).

Animals↗

Hypokalemia downregulates cardiac alpha 1 and skeletal muscle alpha 2 isoforms of Na+,K(+)-ATPase in ferrets.

K+ deficiency decreases amount of Na+,K(+)-ATPase in cardiac and skeletal muscle (SM) and differentially regulates alpha- and beta-subunit isoforms. Expression of the alpha 3 isoform during K+ deficiency is unknown, however. In the myocardium of normal adult ferrets, alpha 1 and alpha 3 isoforms are expressed. Therefore, we examined in ferrets the effects of K+ deficiency in levels of alpha- and beta-subunit isoforms. A K+ deficient diet greatly reduced plasma K+ concentrations. Tissue K+ content in myocardium remained unchanged, whereas that of SM decreased markedly. Corresponding to these changes, Na+,K(+)-ATPase enzyme activity decreased in SM but not in heart. Levels of alpha 1 in heart and alpha 2 in SM decreased, whereas those of alpha 3 in heart and alpha 1 in SM remained unchanged; beta 1 remained unchanged in both tissues. Thus, in ferrets, K+ deficiency differentially regulated the levels of Na+,K(+)-ATPase isoforms in an isoform- and tissue-dependent manner.

Animals↗

Medial preoptic/anterior hypothalamic lesions induce a female-typical profile of sexual partner preference in male ferrets.

In T-maze tests given to gonadectomized ferrets treated daily with estradiol benzoate (EB), females consistently prefer to approach and interact sexually with a stud male whereas male subjects, on average, prefer an estrous female. In the present experiment this sexually allomorphic pattern of partner preference was changed in males given lesions of the medial preoptic area/anterior hypothalamus (mPOA/AH). Electrolytic lesions, which caused extensive bilateral damage to the mPOA/AH, including the sexually dimorphic male nucleus (MN) of the POA/AH, led males to shift their mean preference away from the estrous female to the stud male. Their postoperative profile of partner preference more closely resembled that of sham-operated females than that of sham-operated males or of males which sustained either partial or minimal bilateral damage to the mPOA/AH so as to spare the MN-POA/AH in one or both hemispheres. Males with extensive bilateral mPOA/AH lesions, like sham-operated females, showed an even stronger preference to approach the stud male during T-maze tests in which the subjects could smell, see, and hear the stimulus animals without physically interacting with them. After receiving testosterone propionate, male ferrets with either extensive or partial lesions of the mPOA/AH showed significant deficits in neck gripping and mounting performance in tests with either female or male stimulus animals which were sexually receptive after gonadectomy and EB treatment. The present results, coupled with those of a previous study using excitotoxic mPOA/AH lesions, suggest that the male-typical profile of preference for an estrous female depends on the functional integrity of sexually dimorphic mPOA/AH neurons and the reward engendered by coital interaction with such a female. When these neurons either are destroyed experimentally (as in male ferrets with extensive bilateral mPOA/AH lesions) or are absent (as in sham-operated females), subjects are attracted by distal (possibly chemosensory) incentive cues from a stud male.

Animals↗

Biochemical, mechanical and energetic characterization of right ventricular hypertrophy in the ferret heart.

Ferret right ventricular hypertrophy is characterized by a decreased and prolonged isometric contraction, associated with altered intracellular calcium (Ca2+) regulation. However myofibrillar composition, cross-bridge function and/or energy transfer may also be involved in these contractile disturbances. Therefore, mechanical properties of myofibrils have been studied with Triton X-100-skinned fibres and troponin (Tn) T and I composition has been examined. Mitochondrial function and functional activity of creatine kinase (CK) isoforms have been studied in saponin-skinned fibres of control (C) and hypertrophied (H) ferret right ventricle, to check for a possible mismatch between energy production and utilization. Our results show that neither TnT nor TnI isoform expression, nor myofibrillar Ca2+ responsiveness (similar apparent Ca2+ sensitivity and Hill coefficient) were affected by pressure-overload. Similarly, maximal tension and stiffness, as well as cross-bridge cycling rate (v)--assessed by quick length changes--were not significantly altered. Importantly, passive stiffness was dramatically increased (163 +/- 30 mN/mm2/microns for C v 500 +/- 121 mN/mm2/microns for H; P < 0.02). Moreover, there was a significant correlation between passive stiffness and cross-bridge cycling rate, indicating that a factor involved in the passive stiffness may affect cross-bridge kinetics. Oxidative capacity (normalized to ventricular dry weight), reflecting mitochondrial ATP production and mitochondrial CK efficacy, as well as myofibrillar CK efficacy (assessed by the shift of MgATP-rigor tension curves before and after phosphocreatine addition), were similar in both groups. These results demonstrate that ferret right ventricular pressure-overload was accompanied by a development of myofibrils and a parallel increase of energy production capacity, transfer and utilization. Decreased compliance, probably linked to an increase in the collagen fraction and/or alterations of the cytoskeletal architecture of the overloaded ventricle, could contribute to the slower time course and decreased amplitude of the isometric twitch.

Animals↗

Characterization of neural control and contractile function in airway smooth muscle of the ferret.

Several recent studies have characterized neuroanatomical and neurophysiological aspects of ferret airways, but regional differences in reactivity and cholinergic, adrenergic and non-adrenergic-non-cholinergic (NANC) neural responses have not been examined. The aim of this study was to characterize the contractile and relaxant response elicited by electrical field stimulation (EFS), and the contractions induced by cholinergic agonists in isolated ferret tracheal and bronchial preparations. EFS produced frequency-dependent contractions and relaxations. Contractions in both the trachea and bronchi were inhibited by atropine and potentiated by neostigmine. Tracheal relaxations were found to be entirely adrenergic, but bronchial relaxations were mediated by a combination of adrenergic and inhibitory NANC (i-NANC) innervations. Trachea and bronchi were more sensitive to methacholine (MCh) and carbachol than to acetylcholine (ACh); middle tracheal segments being more sensitive to ACh than distal segments, however, in the presence of neostigmine ACh potency was equal in both segments. The results suggest that regional differences exist in cholinergic responsiveness of ferret trachea and bronchi, resulting from differences in ACh degradation.

Acetylcholine↗

Acrolein-induced smooth muscle hyperresponsiveness and eicosanoid release in excised ferret tracheae.

Acrolein is a ubiquitous toxic air pollutant that can have adverse lung effects. To understand the mechanism governing airway reactivity in relation to acrolein uptake, in vitro experiments were conducted in which excised tracheae from ferrets were exposed for 1 hr to a unidirectional constant flow (100 ml/min) of an acrolein-in-air mixture at several concentrations (0-12.5 ppm). During exposure, acrolein uptake into the trachea was determined by a chromatographic analysis of gas samples taken at the entrance and at the exit of the trachea. Smooth muscle contractility in response to carbachol (CCh), acetylcholine (ACh), and potassium chloride (KCl) was measured following exposure, and eicosanoids released in the perfusate baths were assayed. The results indicate that the fractional uptake into an excised ferret trachea was strongly dependent on inlet concentration, implying that diffusion and reaction processes of acrolein in airway tissue are not linear. Only the low concentration of acrolein caused an increase of eicosanoid release from the exposed tracheae in the perfusate bath; it is possible that, at higher exposure concentration, the epithelium was sloughed off and most of the eicosanoids were lost. Although acrolein did not alter smooth muscle response to KCl, it did increase the contractile responses to CCh and ACh, suggesting an alteration in the pharmacomechanical but not the electromechanical coupling of ferret tracheal smooth muscle; therefore, it is more likely that this hyperresponsiveness occurs primarily by a mobilization of intracellular Ca2+ stores rather than by an increased influx of extracellular Ca2+ through voltage-dependent channels.

Acrolein↗

Acetylcholine and adenosine activate the G protein-gated muscarinic K+ channel in ferret ventricular myocytes.

The properties of the K+ channel activated by acetylcholine (ACh) and adenosine (Ado) were examined in single ferret ventricular myocytes using patch-clamp techniques. In the whole-cell configuration, ACh and Ado induced an inwardly rectifying K+ current and shortened the action potential duration. The effect of ACh was blocked by atropine, while the Ado effect was interrupted by 8-cyclopentyl,1,2-dipropyl xanthine, a specific Ado A1 receptor antagonist. In cell-attached recordings, ACh and Ado added to the pipette solution activated a single population of inwardly rectifying K+ channels, distinct from the iK1 channel. The channel had a slope conductance of approximately 40 pS in symmetrical 150 mM K+ solutions and a mean open time of 0.8 ms. Excision of the patch into the inside-out patch configuration in guanosine triphosphate (GTP)-free solution abolished the channel activity. The channel was reversibly reactivated by adding GTP to the intracellular side of the patch. GTP gamma S activated the channel irreversibly. When the inside-out patch was treated with the A protomer of pertussis toxin (PTX), intracellular GTP no longer activated the K+ channel. The results show that ferret ventricular myocytes possess a K+ channel activated by both muscarinic and Ado A1 receptors. Its electrophysiological properties and the gating by a PTX-sensitive G protein in a membrane-delimited fashion are identical with those of the muscarinic K+ channels in nodal and atrial tissues of other species. In conclusion, the G protein-gated muscarinic K+ channel is expressed in ferret ventricular myocardium and may underlie the direct negative inotropism of ACh and Ado in this tissue.

Acetylcholine↗

Visual response properties and afferents of nucleus of the optic tract in the ferret.

Basic properties of responses to visual stimulation with large moving random dot patterns were studied in ferret nucleus of the optic tract. Retinal input to NOT was assessed by orthodromic electrical stimulation of the optic chiasm and optic nerves. Presence of an input from visual cortex was tested by orthodromic electrical stimulation of ipsilateral area 17. All 51 NOT neurons studied displayed a non-habituating, clearly direction-specific response: discharge rate strongly increased with the stimulus pattern moving horizontally in ipsiversive direction (motion directed towards the recorded hemisphere) and decreased with contraversive stimulus motion. Most latencies to visual stimulation ranged from 80 to 100 ms. Velocity tuning was studied using stimulus velocities between 4 deg/s and 100 deg/s. Discharge rates were most effectively modulated at a stimulus velocity of 20 deg/s. A large portion of the cells studied (91%) could be binocularly activated, although for almost all neurons the contralateral eye was dominant. Through stimulation of the optic chiasm 46 out of 51 NOT neurons could be electrically activated with a latency of 5.42 +/- 0.66 ms (mean +/- SD). For 15 fibers stimulated from both optic chiasm and contralateral optic nerve, conduction velocities between 2.5 and 8.9 m/s, with a mean of 5.1 m/s, were obtained. A major direct input from the ipsilateral retina was not found. Furthermore, 65% of all neurons could be activated through electrical stimulation of visual cortex with a mean latency of 3.7 +/- 1.5 ms, indicating a strong cortical projection to ferret NOT. The functional relevance of response properties of ferret NOT neurons for horizontal optokinetic nystagmus is discussed. Parameters that could be related to formation of a cortico-pretectal projection in mammals are considered.

Afferent Pathways↗

Spatial properties of neurones in the lateral geniculate nucleus of the pigmented ferret.

We used quantitative electrophysiological techniques to study the spatial properties of single units recorded extracellularly in the lateral geniculate and perigeniculate nuclei of the adult pigmented ferret. All neurones examined had approximately circular receptive fields, whose central regions gave responses antagonistic to those elicited from the surrounds. We presented sinusoidally modulated grating patterns, either drifting or counterphased, to obtain spatial frequency tuning curves, contrast sensitivity functions and to assess linearity or non-linearity of each neurone's response. In the ferret, as in other species, two types of lateral geniculate neurone could be distinguished, and we have termed these X-cells and Y-cells; both groups responded briskly to visual stimulation but X-cells gave sustained and linear responses whereas Y-cells responded transiently and non-linearly. Perigeniculate cells gave linear responses. For neurones in the lateral geniculate and perigeniculate nuclei, both the limit of spatial resolution ('acuity') and optimum spatial frequency were inversely related to receptive field eccentricity and the diameter of the receptive field centre. We recorded geniculate neurones in the ferret with acuities up to 3 cycles deg-1 and contrast sensitivities up to 114, values that are lower than those found previously for many geniculate cells in the cat.

Animals↗

Polychlorinated biphenyls (Aroclors 1016 and 1242): effects on survival and reproduction in mink and ferrets.

Diets that contained various levels of supplemental Aroclor 1242 or Aroclor 1016 were fed to mink and ferrets to investigate the chronic toxicity of these PCBs in two closely related species. In mink, Aroclor 1242 was found to be more toxic than comparable or higher levels of Aroclor 1016. The Aroclor 1242 diets caused complete reproductive failure at levels as low as five ppm of the diet. Aroclor 1016 impaired reproduction less than Aroclor 1242. Although fewer females whelped and the four-week kit weights were less than the control animals, no outward signs of abnormalities beyond their smaller size were found in the kits whelped and nursed by dams fed Aroclor 1016. Ferrets were more resistant to the effects of either PCB mixture than were the mink, as noted by the lower mortality rate on the Aroclor 1242 diet and the almost normal level of reproduction on the Aroclor 1016 diet. Feeding Aroclor 1242 at 20 ppm resulted in complete reproductive failure, but was not fatal to adult ferrets. This finding is in sharp contrast to the 100% mortality of adult mink fed the same level. Although the chlorine content is similar in both compounds, Aroclor 1242 has a higher percentage of molecules with five or more chlorines per biphenyl. This difference in higher substituted biphenyl isomer content and/or the reduced levels of contaminants in the Aroclor 1016 mixture may be of major importance in evaluating the toxicity of these compounds.

Animals↗

Identification of a DNA segment in ferret Aleutian disease virus similar to a hypervariable capsid region of mink Aleutian disease parvovirus.

A 401bp DNA fragment of ferret Aleutian disease virus (ADV) was amplified using PCR primers spanning a hypervariable region of mink ADV capsid sequence. The amplified fragment was 88-89% homologous to the same region of previously known sequence of three different strains of mink ADV, however, as low as 54% homology was observed when compared with a 39bp segment known as hypervariable region. Within the predicted 13 amino acid hypervariable region, the ferret ADV sequence differed at 6 positions from the wild type mink Utah1 strain. Three amino acids Gln289, Glu293 and Thr295 in this region were common to the pathogenic ferret ADV, mink Utah1 and ADVK strains, but differed from the cell culture adapted nonpathogenic mink strain ADVG suggesting that these three conserved residues may have some functional significance.

Aleutian Mink Disease Virus↗

Action of a novel drug (Zy 16039) on mucus secretion in the ferret isolated trachea in vitro.

1. The effect of 4-H-2-carboxamido-4-phenyl-thieno-[3,2c]-[1]-benzopyran (Zy 16039) was examined on the smooth muscle contraction, mucus secretion and albumin transudation in the ferret whole trachea in vitro. 2. Zy 16039 (0.1-20 microM) produced a concentration-dependent relaxation of the ferret trachea contracted by methacholine (1 microM) and phenylephrine (10 microM). The relaxations were about 20% of the full contractions. 3. Zy 16039 has no effect on the resting (zero) output of mucus in the ferret trachea. Methacholine-induced mucus secretion was significantly inhibited by Zy 16039, whereas phenylephrine-induced secretion was significantly increased. 4. Methacholine-induced secretion of lysozyme, a marker of serous cell secretion, was inhibited by Zy 16039 both with regard to output and concentration of lysozyme. In contrast, Zy 16039 significantly increased the output of lysozyme due to phenylephrine, with no effect on concentration. 5. Zy 16039 had no significant effect on the rate of output of fluorescent albumin through the tracheal wall. However the concentration of albumin in the mucus samples was changed because of the effect of Zy 16039 on mucus secretion induced by methacholine and phenylephrine. 6. We conclude that Zy 16039 relaxes airway smooth muscle, and either promotes or inhibits mucus secretion depending on its source. It has qualitatively similar actions to vasoactive intestinal peptide.

Albumins↗

Persistence of visna virus in cultured ferret brain cortex and fibroblasts.

Cerebral cortex tissue fragments from newborn ferrets have been cultured in vitro and, when exposed to visna virus, failed to exhibit a cytopathic effect but retained the genome of the virus in a quiescent state for 46 d before synthesizing plaque-forming virus for the subsequent 26 d. In contrast, infection of an established line of cells derived from a ferret's brain with visna virus resulted in the formation of the polykaryocytes typically produced by this virus and a limited synthesis of plaque-forming virus. Ferret cerebral cortex cultures may be suitable in vitro hosts for studies to define the virus-host interactions that result in virus-induced demyelination.

Animals↗

Callosal connections of the ferret primary auditory cortex.

The callosal connections of ferret auditory cortex were studied by making multiple injections of wheat germ agglutinin-horseradish peroxidase into the middle ectosylvian gyrus or by packing crystals of horseradish peroxidase into the transected corpus callosum. The primary area (AI) had strong callosal connections that arose from somata mainly located in layer III. Other layers contained sparsely distributed cells that projected across the midline. The projecting cells occurred over the whole extent of AI but were not homogeneously distributed in layer III. The axons from these cells terminated mainly in the upper layers of the contralateral cortex, where they converged onto three discrete bands. The three elongated bands lay in a dorsoventral orientation, parallel to the tonotopic axis. They were slightly curved and had a fairly uniform width. The posterior band had a width of about 200 microm, while the anterior and middle bands were more variable and had widths of 300-800 microm. The centre-to-centre distance between the posterior and middle bands was 520+/-60 microm and for the anterior to middle bands was 620+/-210 microm. The retrograde labelling produced by the same injections showed that the cell bodies had a higher density in the terminal bands than in the intervening spaces. The bands of dense callosal connections appear to correspond to the binaural summation columns, which have been clearly demonstrated in the ferret, but direct evidence of this will need to be sought in a future study. The discrete nature of the callosal bands in the ferret appears to make it a suitable species for studying the relationship between callosal terminals and those arising in other areas of the brain and for clarifying the possible existence of separate functional systems within the auditory cortex.

Animals↗

Sagittal zonal organization of climbing fibre input to the cerebellar anterior lobe of the ferret.

The organization of climbing fibre input to the cerebellar anterior lobe of the ferret was investigated in barbiturate-anaesthetized animals. Climbing fibre field potentials evoked on electrical stimulation of forelimb and hindlimb nerves were recorded at the cerebellar surface. Based on characteristic latencies of climbing fibre responses and their relative localization along the longitudinal axis of the folia, nine sagittally oriented zones could be distinguished and were tentatively named, from medial to lateral, A, X, B, C1, Cx, C2, C3, D1 and D2. Within the C1, C2 and C3 zones, climbing fibre input from the ipsilateral forelimb was found caudally and from the hindlimb rostrally, while the corresponding topographical representation in the B and D2 zones was medial to lateral. The X, Cx and D1 zones did not receive input from the hindlimb, while input from the forelimb to the A zone was weak. Overall, the sagittal zonal organization of climbing fibre input appears to conform with the compartmentalization of the ferret cerebellum based on the myeloarchitecture of corticonuclear fibres, although the X and Cx zones have not been previously identified. In terms of both general electrophysiological characteristics of input to different zones and intrazonal topographical representation, the organization of climbing fibre input to the ferret cerebellum seems to strongly resemble that in the cat. The findings thus provide evidence of cross-species generality of cerebellar sagittal organization.

Animals↗