Genetic selection on a behavioural fear trait is associated with changes in heart rate variability in quail.

This study investigated whether genetic selection on a divergent behavioural trait of fearfulness (tonic immobility duration) was related to changes in the nervous control of the heart. Quail selected for either long or short tonic immobility (LTI or STI, respectively) duration was compared with an unselected control line (CTI). The autonomic control of the heart was assessed by heart rate variability analysis and pharmacological blockades. Quail were surgically fitted with a telemetric device. Heart rate before injection did not differ between the three lines. The vagal-sympathetic effect (VSE) at rest differed significantly from 1 in CTI and STI quail, suggesting that parasympathetic activity was dominant. In LTI quail, VSE did not differ from 1, suggesting a balance between parasympathetic and sympathetic activities. The intrinsic heart rate reached after the successive injections of propranolol and atropine did not differ between lines and was higher than the heart rate at rest in STI, which was in line with results of VSE at rest. After atropine injection, the sympathetic activity indicated by the low-frequency power was lower in CTI than in the two selected quail. After propranolol injection, the parasympathetic activity indicated by the root of the mean squares of successive differences and the high-frequency power was higher in STI than in CTI and LTI quail. Selection on tonic immobility duration thus appears to be associated with changes in the sympathovagal control of the heart, which may influence behavioural responses to stressful situations.

Emotional reactivity modulates autonomic responses to an acoustic challenge in quail.

Emotional reactivity modulates autonomic responses to an acoustic challenge in quail. Physio Behav 00(0) 000-000, 2006. This study investigated the relationship between emotional reactivity and behavioral and autonomic responses to an acoustic stimulus in quail. It was hypothesized that birds with high emotional reactivity would have higher motor inhibition combined with higher sympathetic activation than birds with low emotional reactivity. Two experiments were performed. The first looked for correlations between emotional reactivity, evaluated by a tonic immobility test, and motor and Heart Rate Variability in relation to an acoustic stimulus. The second experiment compared the motor and autonomic responses to the acoustic stimulus of quail selected on either long (LTI) or short (STI) duration of tonic immobility. The first experiment showed that the acoustic stimulation induced motor inhibition and cardiac activation. Correlations were found between tonic immobility duration and both autonomic activity before stimulation and sympathovagal balance after stimulation. In the second experiment, LTI quail showed strong sympathetic activation, whereas STI quail showed parasympathetic and sympathetic activation. The activation of the parasympathetic system induced by the noise in STI quail can be explained by the predominance of this system at rest in this line. In conclusion, both the basal autonomic activity and the autonomic responses differed according to the emotional reactivity, and changes in autonomic activity appear to be related to the genetic selection process.

Does nutritive and non-nutritive sucking reduce other oral behaviors and stimulate rest in calves?

After a milk meal, bucket-fed calves show non-nutritive oral activities, including cross-sucking, and this can discourage producers from rearing them in groups. Sucking is known to induce a quiet state in humans and rats. We examined if nutritive sucking affects non-nutritive oral activities in calves, if it reduces arousal (assessed through behavior and cardiac activity), and if sucking a dry teat can compensate for the lack of nutritive sucking. In Exp. 1, the behavior and the cardiac activity of individually housed calves fed milk from a bucket were compared to those of calves fed milk through a teat. During the meal, the heart rate of bucket-fed calves was higher than that of teat-fed calves (P < 0.0001). After the meal, only bucket-fed calves displayed bar sucking. Compared to the teat-fed calves, they spent more time licking their pen or their neighbor (P < 0.001 and P < 0.05), their heart rate was less variable (P < 0.01), and they lay down with the head unsupported by the neck less quickly (latency to lie down: 51 min vs 42 min, P < 0.05). In Exp. 2, individually-housed bucket-fed and teat-fed calves were observed with or without access to a non-nutritive teat after the meal. Bucket-fed calves sucked the dry teat for longer than teat-fed calves (P < 0.001). In bucket-fed calves, access to the dry teat reduced the time spent nibbling (P < 0.01) and tended to shorten the latency to lie down (P < 0.10). In Exp. 3, group-housed bucket-fed calves were compared with group-housed calves fed with an automatic teat feeder system. Bucket-fed calves spent more time nibbling at 1 mo, but at 3 mo they spent less time nibbling and cross-sucking; they drank more milk and put on more weight. We conclude that, for calves housed individually, teat-feeding reduces non-nutritive oral activities after the meal and induces a calmer state than bucket-feeding. Providing calves with a dry teat partly compensates for the lack of nutritive sucking. For calves housed in groups, the use of an automatic teat feeder may not reduce calves' motivation for sucking. No improvement of growth was observed with teat-feeding either with a teat-bucket or with an automatic feeder.

Effect of autonomic blockers on heart period variability in calves: evaluation of the sympathovagal balance.

This study was designed to validate the measures of heart period variability for assessing of autonomic nervous system control in calves. Eight calves received an injection of either 0.5 mg/kg atenolol (sympathetic tone blockade), 0.2 mg/kg atropine sulfate (parasympathetic tone blockade), 0.5 mg/kg atenolol + 0.2 mg/kg atropine sulfate (double autonomic blockade) or saline. In the time-domain, we calculated the mean instantaneous heart rate (HR), mean of RR intervals (MeanRR), standard deviation of RR intervals (SDRR) and that of the difference between adjacent intervals (RMSSD). In the frequency-domain, the power of the spectral band 0-1 Hz (TPW), the power of the 0-0.15 Hz band (LF), that of the 0.15-1 Hz band (HF), and the LF/HF ratio were considered. The net vago-sympathetic effect (VSE) was calculated as the ratio of MeanRR in a defined situation to MeanRR during the double blockade. Atenolol injection had no effect on cardiac activity, whereas atropine induced large modifications which were moderated when atenolol was administered at the same time. VSE, HR, MeanRR and RMSSD were found to be valid indicators of the parasympathetic tone of calves because of large variations due to the drug and low individual variations. No measure reflected the sympathetic tone.

Discrimination between shepherds by lambs reared under artificial conditions.

We studied the ability of 32 lambs reared artificially in groups of four to discriminate between their shepherd and an unknown shepherd. Half of the lambs were bottle fed in isolation by one shepherd during the first 3 wk. The other half was fed alternately by three shepherds. Lambs had no visual contact with humans for the next 3 wk. Lambs were weaned at 6 wk of age and reared together with the minimum human contact necessary for rearing management. Lambs were tested at 3, 6, and 14 wk of age, investigating the effect of the rearing conditions on the response to isolation and to reunion with the known or an unknown shepherd. During tests, lambs were observed 1) in isolation for 1 min, 2) in the presence of a shepherd who entered and squatted at one end of the pen for 1 min, trying to touch the lambs if they approached, 3) again in isolation for 1 min. Early rearing management (one vs three shepherds) had no significant effect on any criteria studied. Lambs vocalized and moved less when in the presence of the shepherd than when isolated. They vocalized less, moved less, approached more quickly, and interacted more with the known than with an unknown shepherd. The difference persisted after 3 wk spent without visual human contact. However, no difference was evident at 14 wk of age. The effect of shepherd knowledge is clearly demonstrated by this experiment after an intensive early period of contact.

Distribution of glial fibrillary acidic protein (GFAP) in the cochlear nucleus of adult and aged rats.

The age-related change in glial fibrillary acidic protein (GFAP) immunoreactivity was analyzed in young (3 months) and old (24 months) adult rat cochlear nuclei (CN). Quantitative analyses show a significant increase with age, in the number of GFAP positive astrocytes and processes in the old adult when compared with the young adult rat. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats. The increase in GFAP immunoreactivity was related to an increase of both GFAP positive astrocyte number and processes. The increase of GFAP positive astrocytes may be due either to an alteration of auditory nerve fibers, changing the trophic interactions with post-synaptic cells, or to intrinsic alterations of CN neurons and local circuits reflecting aging of the CN.

N-Methyl-D-aspartate receptor subunits NR1 and NR2C are overexpressed in the inferior colliculus of audiogenic mice.

Some non-DBA2 Albino Swiss mice exhibit noise induced epileptic seizures during a short period of postnatal development. Because N-methyl-D-aspartate (NMDA) glutamate ionotropic receptors are involved in the occurrence of audiogenic seizures, we investigated by in situ hybridization methods, the expression of the different subunits (NR1, NR2A, NR2B, NR2C) of this receptor in the central nucleus of the inferior colliculus (IC), a main relay of the auditory pathways. At postnatal day 20, the NR2C subunit is highly expressed in the IC of convulsive mice, while in non-convulsive mice a slight signal is only found for NR1, NR2A, and NR2B. In adult mice, the NR1 and NR2A signals are observed while the NR2B signal is almost undetectable. The audiogenic susceptibility may be related to the transient expression of the NR2C subunit during a brief neonatal period during which synaptic reorganization happens.

Histochemical and scanning electron microscopic studies of supernumerary hair cells in embryonic rat cochlea in vitro.

In the embryonic organ of Corti supernumerary hair cells were observed when developed in organotypic cultures. Hair cells ranging in up to two rows of inner hair cells (IHCs) and up to nine rows of outer hair cells (OHCs), were observed by phalloidin histochemistry. The total number of hair cells may double in some explanted cochleae compared to control ones. Cuticular plates of hair cells displayed an actin-free zone corresponding to the kinocilium location, differently located and indicating different degrees of differentiation and maturation. Moreover, some hair cells had a small apical surface area and a centrally located kinocilium, revealing immaturity. Under scanning electron microscopy, stereocilia appeared to differentiate normally, as compared to the in vivo development. The staircase pattern of the stereociliary bundles was reached on most of the hair cells with a 'V' shape on the OHCs and hemispherical one on the IHCs. Hair cell polarity was not homogeneous along the length of the tissue. Organs of Corti explanted at birth developed a weaker number of supernumerary hair cells showing a decrease of supernumerary hair cells with the developmental stage of the explant. These results provide evidence for supernumerary hair cells in the mammalian cochlea in culture, without loss or injury to preexisting hair cells.

Distribution of cytoskeletal proteins (neurofilaments, peripherin and MAP-tau) in the cochlea of the human fetus.

We report here an immunohistochemical study of the distribution of intermediate filaments (neurofilament, peripherin) and a microtubule-associated protein, tau, in the human fetal cochlea at 27 weeks of gestation. Neurofilament immunoreactivity (160 and 200 KDa) was localized in afferent and efferent fibers of the cochlear innervation and restricted to a few small spiral ganglion neurons. Peripherin immunoreactivity was specifically distributed in some small ganglion neurons and in their central and peripheral extensions, particularly in fibers reaching the lower part of the outer hair cells. Double immuno-labelling studies with these neurofilaments and peripherin antibodies show that only small neuron cell bodies were stained. Morpholometrical data indicate that immunostained neurons could be related to the Type II neuron population in the spiral ganglion. Tau protein was localized in intraganglionic spiral bundle fibers and in fibers that reach the lower part of hair cells. These observations suggest that neurofilament and peripherin antibodies stain a particular population of human spiral ganglion neurons with Type II characteristics. Moreover, the specificity of peripherin labelling in Type II cells and their processes suggest that peripherin could be used as a probe for the developmental study of this system in the human cochlea. On the other hand, tau antibody appeared as a marker for efferent fibers during development and could give information on the ontogenesis of efferent innervation.

Distribution and metabolism patterns of plasma 7S- and beta-NGF in the adult male rat.

There is a great deal of controversy on the existence of NGF in body fluids and tissues. To date it remains unknown whether this peptide accumulates preferentially at significant levels in different organs. Thus we undertook the evaluation of kinetic parameters of the disappearance of blood of 125I-7S-NGF and 125I-beta-NGF after intravenous injection in male adult rats. Our results indicate that the plasma half-life of 125I-7S-NGF is approximately twice as long as for 125I-beta-NGF (respectively 61.7 +/- 11.7 min and 36.3 +/- 2.2 min) while the distribution volume is not significantly different between both peptides. Furthermore, the uptake of radioactive NGF by different tissues seems very low as shown by 125I-7S-NGF and 125I-beta-NGF content of the sampled organs compared to the plasma concentration at the same time. These results indicate that the tissue uptake of circulating 7S and beta-NGF is very low in the adult rat. Thus in these animals NGF did not cross the blood-brain barrier and did not accumulate in peripheral organs which are known to contain subsequent amounts of this peptide. This lack of deposition might be due to a binding with plasma proteins (probably alpha 2-macroglobulin).

Neurotrophins and the development of cochlear innervation.

The rapid progress in the past few years concerning neurotrophic factor research, has greatly stimulated advances in developmental neurobiology of hearing. We have summarized evidence that neurotrophins are expressed by auditory sensory epithelia during the time at which ganglion cells with neurotrophin receptors send their processes to these epithelia. Recent findings have led to the identification of BDNF and NT3 as responsible substances. Since no NGF mRNA nor the NGF high affinity receptor component trkA mRNA were detectable during the development of cochlear structures, this factor is not likely to be an important neurotrophin at this level. By their biological activity, neurotrophins could be responsible for chemotrophic, differentiation, survival, and maintenance functions at the afferent as well as at the efferent level of the inner ear development.

Hair cell overproduction in the developing mammalian cochlea in culture.

Hair cells transduce acoustics into electrical signals that are conveyed to the brain by auditory nerve fibres. Hair cells loss in mammals due to ageing, ototoxic drugs or noise, leads to irreversible hearing impairment. One objective would be to replace lost cells by regeneration or production of new hair cells. We report an overproduction of hair cells in the developing cochlea of the rat in culture without adding drugs, without previous injury or special manipulations of the explants. The overproduction of hair cells does not depend on the culture medium or on the innervation of the organ of Corti. Younger foetal explants show higher potency for the production of supplementary hair cells than older ones. This is the first report of the generation of extra hair cells in mammals without previous hair cell loss or treatment with drugs.

Ontogenesis of type II spiral ganglion neurons during development: peripherin immunohistochemistry.

In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.

Basic FGF-like protein in the lower stato-acoustic system of the neonatal and adult rat.

Basic fibroblast growth factor (bFGF)-like localization was studied immunohistochemically in the lower auditory tract of neonatal and adult rats. During the neonatal period, bFGF-like immunoreactivity is present in the cytoplasm of inner hair cells, spiral ganglion cells, Scarpa's ganglion cells, in auditory brain stem nuclei and in vestibular nuclei. At the adult stage, bFGF-like protein is widely distributed in the auditory brain stem but was not found in the cochlea. These results suggest that bFGF could be implicated in the development as well as in the neuronal maintenance and plasticity of the auditory system.

Expression of the beta-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig.

Steady-state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGF mRNAs was in the order vas deferens greater than epididymis greater than or equal to seminal vesicles much greater than testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF-like and proNGF-like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis.

Immunohistochemical localization of nerve growth factor receptor in the cochlea and in the brainstem of the perinatal rat.

Nerve growth factor receptor (NGF-R) localization was studied immunohistochemically in the cochlea and in the brainstem of the perinatal rat, using a specific monoclonal antibody directed against the rat NGF-R. In the cochlea, NGF-R immunoreactivity is positive during the whole perinatal period studied, and is located at the hair cell level, in fibers that reach the organ of Corti, in the intraganglionic spiral bundle and in some small bundles of fibers in the auditory nerve. In the brainstem, NGF-R is detected in auditory structures such as the ventral cochlear nucleus, the superior olivary complex, the nuclei of the trapezoid body and the trapezoid body. Many auditory structures labelled by the NGF-R antibody are implicated in the efferent cochlear innervation. These results suggest that NGF could be implicated in interactions between auditory receptors and efferent innervation of the developing cochlea. This coincides with findings on the immunohistochemical localization of NGF-like protein in the organ of Corti of the developing rat. Moreover, these observations could be related to an early prenatal development of auditory efferent innervation.

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins.

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.

Immunocytochemical localization of neurofilament protein subunits in the spiral ganglion of the adult rat.

Spiral ganglion neurons from adult rats were treated with several monoclonal antibodies that react with neurofilaments (NFs) and NF subunits. An antibody against NFs used with immunocytochemical techniques showed a strong reaction with most neuron processes in the spiral ganglion, whereas only a few neurons presented a reaction. Using monoclonal antibodies against the 3 subunits, we obtained the same results with a small percentage of neurons labelled. From quantitative observations, reacting neurons showed the same percentage as and a smaller size than T II neurons observed with a more conventional method. This shows that reacting neurons are indeed T II neurons and that they can easily be differentiated by an accumulation of NFs in their perikaryon by well characterized commercially available antibodies.

Immunohistochemical localisation of nerve growth factor-like protein in the organ of Corti of the developing rat.

The presence of nerve growth factor (NGF)-like protein was investigated in the cochlea of the developing rat between birth and postnatal day 30, by the indirect immunofluorescence technique. Nerve growth factor-like protein could be detected from birth up to day 8. The immunostaining was localised within the hair mainly above their nuclei. No NGF-like immunoreactivity was observed in spiral ganglion cells. The data suggest that NGF acts as a neurotrophic factor, especially for efferent endings in the developing cochlea.