Efferent innervation of turtle semicircular canal cristae: comparisons with bird and mouse.

In the vestibular periphery of nearly every vertebrate, cholinergic vestibular efferent neurons give rise to numerous presynaptic varicosities that target hair cells and afferent processes in the sensory neuroepithelium. Although pharmacological studies have described the postsynaptic actions of vestibular efferent stimulation in several species, characterization of efferent innervation patterns and the relative distribution of efferent varicosities among hair cells and afferents are also integral to understanding how efferent synapses operate. Vestibular efferent markers, however, have not been well characterized in the turtle, one of the animal models used by our laboratory. Here we sought to identify reliable efferent neuronal markers in the vestibular periphery of turtle, to use these markers to understand how efferent synapses are organized, and to compare efferent neuronal labeling patterns in turtle with two other amniotes using some of the same markers. Efferent fibers and varicosities were visualized in the semicircular canal of red-eared turtles (Trachemys scripta elegans), zebra finches (Taeniopygia guttata), and mice (Mus musculus) utilizing fluorescent immunohistochemistry with antibodies against choline acetyltransferase (ChAT). Vestibular hair cells and afferents were counterstained using antibodies to myosin VIIa and calretinin. In all species, ChAT labeled a population of small diameter fibers giving rise to numerous spherical varicosities abutting type II hair cells and afferent processes. That these ChAT-positive varicosities represent presynaptic release sites were demonstrated by colabeling with antibodies against the synaptic vesicle proteins synapsin I, SV2, or syntaxin and the neuropeptide calcitonin gene-related peptide. Comparisons of efferent innervation patterns among the three species are discussed.

Auditory physiology and anatomy of octavolateral efferent neurons in a teleost fish.

Vertebrate hair cell systems receive innervation from efferent neurons in the brain. Here we report the responses of octavolateral efferent neurons that innervate the inner ear and lateral lines in a teleost fish, Dormitator latifrons, to directional linear accelerations, and compare them with the afferent responses from the saccule, the main auditory organ in the inner ear of this species. Efferent neurons responded to acoustic stimuli, but had significantly different response properties than saccular afferents. The efferents produced uniform, omnidirectional responses with no phase-locking. Evoked spike rates increased monotonically with stimulus intensity. Efferents were more broadly tuned and responsive to lower frequencies than saccular afferents, and efferent modulation of the otolithic organs and lateral lines is likely more pronounced at lower frequencies. The efferents had wide dynamic ranges, shallow rate-level function slopes, and low maximum discharge rates. These findings support the role of the efferent innervation of the otolithic organs as part of a general arousal system that modulates overall sensitivity of the peripheral octavolateral organs. In addition, efferent feedback may help unmask biologically relevant directional stimuli, such as those emitted by a predator, prey, or conspecific, by reducing sensitivity of the auditory system to omnidirectional ambient noise.

In vivo dynamic ME-MRI reveals differential functional responses of RA- and area X-projecting neurons in the HVC of canaries exposed to conspecific song.

HVC (nidopallial area, formerly known as hyperstriatum ventrale pars caudalis), a key centre for song control in oscines, responds in a selective manner to conspecific songs as indicated by electrophysiology. However, immediate-early gene induction cannot be detected in this nucleus following song stimulation. HVC contains neurons projecting either towards the nucleus robustus archistriatalis (RA; motor pathway) or area X (anterior forebrain pathway). Both RA- and area X-projecting cells show auditory responses. The present study analysed these responses separately in the two types of HVC projection neurons of canaries by a new in vivo approach using manganese as a calcium analogue which can be transported anterogradely and used as a paramagnetic contrast agent for magnetic resonance imaging (MRI). Manganese was stereotaxically injected into HVC and taken up by HVC neurons. The anterograde axonal transport of manganese from HVC to RA and area X was then followed by MRI during approximately 8 h and changes in signal intensity in these targets were fitted to sigmoid functions. Data comparing birds exposed or not to conspecific songs revealed that song stimulation specifically affected the activity of the two types of HVC projection neurons (increase in the sigmoid slope in RA and in its maximum signal intensity in area X). Dynamic manganese-enhanced MRI thus allows assessment of the functional state of specific neuronal populations in the song system of living canaries in a manner reminiscent of functional MRI (but with higher resolution) or of 2-deoxyglucose autoradiography (but in living subjects).

Morphology and monoaminergic modulation of Crustacean Hyperglycemic Hormone-like immunoreactive neurons in the lobster nervous system.

Neuronal somata located near branch points in the second thoracic nerve roots of the lobster are immunoreactive for Crustacean Hyperglycemic Hormone (CHH)-like peptides, a family of putative stress hormones. We have employed intracellular dye injection, immunostaining, and confocal imaging to observe the anatomy of these root neurons, which are morphologically diverse and dye coupled. Some root neurons contribute to neurosecretory structures at the points of exit of the root from the nerve cord. Other CNS-projecting root neurons send projections into the T5-A1 interganglionic connectives. Neurosecretory elements of the serotonin (5HT) and octopamine (OCT) systems, implicated in postural control and aggression, terminate densely in the vicinity of the second thoracic root neurons. We have confirmed by double immunostaining for 5HT and CHH-like peptides that the endings of the 5HT neurons are in close apposition to root neurons in the superficial regions of the root. We have also extended previous studies documenting electrophysiological responses of the root neurons to 5HT or OCT. Bath-applied 5HT and OCT inhibit the spontaneous bursting activity of root neurons at concentrations higher than 100 nM. The root neurons desensitize to the persistent presence of high concentrations of 5HT, but not OCT, in the bath. Nanomolar concentrations of OCT, but not 5HT have an excitatory effect on the spontaneous bursting activity of root neurons. This region of the lobster nervous system is of continuing interest, as identified neurons of three neuromodulatory systems implicated in stress and aggression converge and interact at the level of identified neurons.

Biotinylated dextran amine as a marker for fetal hypothalamic homografts and their efferents.

We have explored the use of biotinylated dextran amine (BDA) as a marker for labeling fetal brain grafts and their connections with the host. As a model system we used transplantation of the hamster suprachiasmatic nucleus, the site of an endogenous biological clock governing circadian rhythms. Similar transplants into arrhythmic hosts have been shown to restore behavioral function with a period specific to the donor. For locomotor rhythms, efferent connections are not necessary. For other responses, including endocrine rhythms, efferent connections may be necessary. In order to visualize homografts and their efferents, injections of BDA, an anterograde tracer, were made into the anterior hypothalamic (AH) region containing the SCN or into the dorsal cortex (CTX) of fetal hamster brains. The fetal AH or CTX was microdissected out and stereotaxically implanted into the third ventricle of intact, adult hamsters. After 2, 4, 8, or 12 weeks, hosts were sacrificed and their brains were processed for detection of BDA by either histochemistry or immunofluorescence. BDA intensely labeled graft neurons, their dendrites, and axons with minimal or no spread to the adjacent host brain. Labeled graft axons could be followed for long distances (>1 mm) into the host brain and graft-derived varicosities formed close contacts with host neurons. BDA-labeled graft neurons, located at the perimeter of the graft, also extended dendrite-like processes into the host parenchyma. We conclude that BDA is a useful marker for fetal homografts and their efferents for survival times of less than 2 months.

Projections of the mediolateral part of the lateral septum to the hypothalamus, revealed by Fos expression and axonal tracing in rats.

The lateral septum participates in a variety of functions involving the hypothalamus. The present study investigated the effect of an electrical stimulation of the mediolateral part of the lateral septum on the expression of Fos in the hypothalamic nuclei by using immunohistochemical methods in anaesthetised and free-moving rats. We analysed in another series of rats the direct projections of the lateral septum by axonal anterograde tracing with biotinylated dextran-amine. Tracing was used in combination with Fos labelling in a third series of animals. Stimulation induced an expression of Fos in neurones located in anteroventral and anterodorsal preoptic nuclei, medial preoptic area, anterior hypothalamic nucleus, subparaventricular zone, dorsomedial nucleus, lateral hypothalamic area and mammillary nucleus. The distribution of Fos-immunoreactive neurones conforms to the topographic organisation of direct projections from the lateral septum, as revealed by axonal tracing. These results suggest that the lateral septum activates definite hypothalamic structures by a direct link. Some structures displayed substantial Fos labelling whereas they received a slight, or no projection, from the lateral septum. This was particularly evident in the core of the ventromedial nucleus and in areas known to contain tubero-infundibular neurones. This observation suggests that the lateral septum may also exert an indirect control, via polysynaptic links, on hypothalamic structures including nuclei involved in neuroendocrine mechanisms.

The effect of contralateral broad-band noise on acoustic distortion products from the human ear.

In the first part of the study, the effect of ipsilateral stimulus level on contralateral suppression of the 2f2-f2 distortion product by broad-band noise at 60 dB SPL RMS is examined. The levels of the primary stimuli were manipulated independently, giving f1 and f2 growth curves at four different f2 frequencies for four subjects. These typically bell-shaped, growth curves are shifted vertically to lower distortion levels and, in some cases, horizontally to higher primary stimulus levels. These results can be interpreted as an attenuation of both the primary stimuli and the distortion product and could be caused by simple acoustic attenuation produced by middle ear muscle activity, efferent activity or a combination of the two. In the second part of the study, the same contralateral stimulus was used while measuring both 3f1-2f2 and 2f1-f2 distortion products from the ipsilateral ear. The frequency separation of the primary tones was varied. This produced an approximately bandpass shape with the level peaking when the distortion frequency was approximately half an octave below f2, as previously described (Brown and Gaskill, 1990). This shape is thought to be linked with frequency selectivity in the cochlea. Contralateral broadband noise did not affect the tuning or the centre frequency of the bandpass shape or the mean group delay. It did reduce the size of the distortion peak and, in particular, it affected the peak-to-trough height of the 'fine structure' in the amplitude. Vector analysis revealed that the fine structure was due to a signal with substantial delay (probably from the distortion product 'place') which was summed with a larger, less delayed component (probably directly from the f2 'place'). The greater effect of contralateral stimulation on the more delayed component may reflect differences in efferent effect with complex (stimulus place), rather than simple (distortion product place) stimuli.

The course and distribution of medial efferent fibers in the cochlea of the mustached bat.

The course and distribution of medial olivocochlear (MOC) nerve fibers were studied in the cochlea of the mustached bat. This animal is of interest because of the very sharp tuning of the ear and fine frequency resolution in small frequency bands near 60 and 90 kHz. The MOC fibers arise from about 400 cells in the dorsomedial periolivary (DMPO) nucleus and they are distributed to approximately 4500 outer hair cells (OHCs), resulting in an average OHC unit size of 11.25. Individual fibers appear to have a small number of branches and each branch entering the tunnel of Corti terminates on a patch of OHCs. The patch size is typically 1-3 OHCs with the smallest average patch sizes in the regions tuned to 60 and 90 kHz. The majority of the MOC terminals are derived from the contralateral DMPO. Contralateral vs. ipsilateral projecting fibers are not preferentially distributed within any of the three rows of OHCs or within specific regions throughout most of the cochlea. It can be concluded that the main differences between the mustached bat's MOC system and that of most other mammals are: (1) origin from a single nucleus; (2) relatively small sizes of the patches; (3) a single terminal on each OHC; (4) a gradient in the size of the terminals but not in the number of terminals from row to row or from base to apex.

Electrical stimulation activates two different sites within the guinea pig cochlea.

This study investigates whether auditory brainstem responses (ABRs) can be used to assess the functioning of electrically stimulated cochleas. Electrically evoked auditory brainstem responses (EABRs) were recorded in guinea pigs with normal hearing and guinea pigs deafened by amikacin, a powerful ototoxic antibiotic, combined with diuretic aminooxyacetic acid (AOAA). Two different types of EABRs were observed in normal animals, depending on the electrical pulse intensity applied to the round window: long-latency brainstem responses were evoked by low stimulation intensities, short-latency brainstem responses by high intensities. The absence of effect of strychnine applied intracochlearly ruled out the possibility of medial efferents being involved in these responses. Conversely, an intracochlear application of tetrodotoxin (TTX), an Na(+)-channel blocker, resulted in the disappearance of both types of responses, attesting that the sites activated by the electrical stimulation were located within the cochlea. In AOAA/ amikacin poisoned cochleas, in which most of the hair cells were missing with apparently normal ganglion neurons, the long-latency brainstem responses evoked by low intensities were completely lacking. These findings suggest that low currents applied to the round window of the guinea pig cochlea primarily activate the hair cells, the neurons being directly excited at higher intensities.

Evoked otoacoustic emissions and auditory selective attention.

The auditory system has an extensive peripheral efferent innervation. The question addressed in this paper is whether the olivocochlear bundle (OCB) efferent system innervating the outer hair cells (OHC) of the cochlea plays a role in selective attention. As evoked otoacoustic emissions (EOAE) provide a measure of the active micromechanical properties of OHCs, they can be used to assess the role of the efferent system in attention. Six experiments using tone-pip EOAEs are reported. In each experiment, EOAEs generated by 1 or 2 kHz tone pips when they were attended were compared with EOAEs to the same stimuli when they were unattended. In three experiments (1-4), a non-linear stimulus difference method was used to record a pure cochlear component of EOAEs. In Exps. 1-5, 1 and 2 kHz tone pips were delivered to the same ear and the difficulty of the subjects' task was manipulated in order to produce a more focussed attentional state or contralateral noise was presented to determine whether attention effects are dependent upon having an already activated efferent system. In Exp.6, the 1 and 2 kHz stimuli were delivered to opposite ears. A total of 70 subjects participated in the six experiments. There were no effects of attention on EOAEs in any of the experiments in the direction of previously reported effects. The results of these first six experiments employing simple attention switches between fixed auditory objects do not support active cochlear involvement in selective attention.

The influence of evoking stimulus level on the neural suppression of transient evoked otoacoustic emissions.

This study concerns the suppression of transient evoked otoacoustic emissions (TEOAEs) by contralateral noise. The suppression is interpreted as neurally induced changes in cochlear mechanics. The magnitude of TEOAE suppression is explored in response to a single level of contralateral noise, in 20 normal subjects, and as a function of TEOAE evoking stimulus power in 6 subjects. TEOAE were found to be relatively more susceptible to contralateral suppression when the TEOAE evoking stimulus was low. This suggests that saturation of the TEOAE generator by the evoking stimulus reduces the susceptibility of the generator to neural suppression. However, this relation did not hold between ears. Those ears in which the TEOAE seemed easier to saturate were easier to suppress by contralateral noise. We have concluded that TEOAE generators can differ in their susceptibility to neural suppression. Ears in which the TEOAE generating mechanism is less dependent on the ipsilateral evoking stimuli power level, are also naturally more susceptible to efferent suppression.

Regenerated nerve fibers in the noise-damaged chinchilla cochlea are not efferent.

Nerve-fiber regeneration in the chinchilla cochlea following a traumatic noise exposure was systematically described by Bohne and Harding (1992). However, their study did not determine the origin of the regenerated nerve fibers (RNFs). In the present study, 23 chinchillas were exposed for 12 h to a 0.5 kHz octave band of noise at 120 dB SPL. After a 3-month or 1-year recovery period, their right cochleas were incubated to demonstrate acetylcholinesterase (AChE) activity and then briefly counterstained with Neutral Red or OsO4. Their left cochleas were fixed with OsO4 and dissected using a combined organ of Corti (OC)/modiolus technique that preserved both structures for high-resolution microscopy. All cochleas were prepared as plastic-embedded flat preparations. Damage was located in the basal two-thirds of the cochlea and generally consisted of multiple lesions in the OC, often involving total degeneration of one or more OC segments (i.e., OC wipeouts). The OC wipeouts were separated from one another by areas which contained some identifiable cells of the OC (i.e., OC remnants). Most RNFs were found in OC wipeouts adjacent to OC remnants. In those animals (83%) with significant OC damage, 13 (100%) 3-month-recovery chinchillas had 1-96 RNFs while 6 (86%) 1-year-recovery chinchillas had 7-62 RNFs. In the AChE-stained cochleas, none of the RNFs were AChE-positive, but normal AChE-positive fibers were found in the undamaged apical turn. A variable number of surviving spiral ganglion cells was present in those regions of Rosenthal's canal that had originally innervated the missing hair cells in the OC wipeouts and remnants. It is concluded that RNFs are not part of the efferent cochlear system and therefore, most likely belong to the afferent system.

Suppression of stimulus frequency otoacoustic emissions by contralateral noise.

The effect of different bands of contralaterally presented noise at low and moderate intensities on stimulus frequency otoacoustic emissions (SFOAE) from human ears is examined. A SFOAE evoked by a continuous stimulus tone and suppressed by a second tone to produce an SFOAE residual was chosen as the probe to determine the effect of the efferent input. At low levels of contralateral noise, a band centred on the ipsilateral stimulus frequency was the most effective suppressor of the SFOAE residual. For higher levels of the contralateral stimulus, noise bands containing higher frequency components produced most reductions in the SFOAE residual. Small changes in the phase of the SFOAE residual during the contralateral noise were also recorded. Increases in the SFOAE residual onset latency were also found to be small, being around 1 ms. In some cases increases in the level of the SFOAE residual produced by low-frequency suppressors were recorded during the contralateral noise presentation. The results are discussed in the context of current knowledge of the functioning of the auditory efferent innervation, and it is suggested that the method of evoking SFOAEs presents a viable method for determining the effect of efferent stimulation on cochlear mechanics which also allows possible artifact contamination to be readily identified.

Binaural noise suppresses linear click-evoked otoacoustic emissions more than ipsilateral or contralateral noise.

We studied the efferent suppression of click-evoked otoacoustic emissions with 65 dB SPL of white noise presented to left, right, or sometimes both, ears for 408 ms. Each burst of noise preceded a series of four unipolar 80 microseconds 65 dB peak Sound Pressure clicks, presented to the left ear only. The first click of the four-click group followed the end of the noise by either 1, 2, 5, 10, 20, 50, 100 or 200 ms; each subsequent click was offset by 20 additional ms via an ILO88 system with special programming modifications. Conditions were alternated so that a 'without noise' condition preceded a 'with noise' condition for three repetitions of 600 clicks per trial. Seven subjects with normal hearing participated in the study, and three of the seven participated in a test-retest reliability study. Results showed the greatest suppression followed binaural stimulation ending within one to five ms of the first click in the pulse train. Somewhat less suppression was seen following ipsilateral stimulation. The least amount of suppression was seen following contralateral stimulation, suggesting that previous research using contralateral stimulation may underestimate efferent effects. We saw no effects when the end of the noise was 100 ms or more away from the beginning of the click train.

Retinofugal and retinopetal projections in the cichlid fish Astronotus ocellatus.

The retinofugal and retinopetal projections of the cichlid fish Astronotus ocellatus were studied by applying cobaltous-lysine to the optic nerve. Retinal axons terminate bilaterally in a preoptic-suprachiasmatic region between the base of the third ventricle and the anterior genu of the horizontal commissure and among periventricular cells along the sides of the ventricle. Other retinal axons innervate the tuberal region of the hypothalamus anterior to the infundibulum. Targets innervated in the pretectum include nucleus lateralis geniculatus and dorsal, medial, and ventral pretectal nuclei. Three other targets (nucleus opticus dorsolateralis, nucleus opticus commissurae posterior, nucleus opticus ventrolateralis) are innervated by fibers that leave the medial edge of the dorsal optic tract. Two other targets (basal optic nucleus and accessory optic nucleus) are innervated by fibers from the ventral optic tract. These retinal projections are similar to those previously reported for goldfish in an experiment that used the cobaltous-lysine method (Springer and Gaffney, J. Comp. Neurol. 203:401-424, '81). Retinotectal optic axons were found in a superficial lamina just above the stratum opticum, in the stratum opticum, in three layers of the stratum fibrosum et griseum superficiale, in a lamina just beneath the stratum fibrosum et griseum superficiale, and in the stratum album centrale just above the stratum periventriculare. This result is similar to that previously reported for goldfish; however, the spatial relationships between the various retinorecipient laminae differ for goldfish and Astronotus ocellatus. Efferents to the retina originate in two nuclei and both project contralaterally. The first is the nucleus olfactoretinalis, which is located ventrally between the olfactory lobe and telencephalon. It consists of about 400 cells, of which, approximately 200 cells project to the retina. The second retinopetal nucleus, nucleus thalamoretinalis, is a diffuse group of about 200 cells that project to the retina.

Efferent systems of the rabbit visual cortex: laminar distribution of the cells of origin, axonal conduction velocities, and identification of axonal branches.

Several efferent systems of visual area I in Dutch rabbits were studied with anatomical (horseradish peroxidase) and physiological (antidromic) methods. Anatomical studies provided information regarding the laminar origin of the projections to the contralateral hemisphere, visual area II, the dorsal lateral geniculate nucleus, and the superior colliculus. Physiological studies provided information regarding conduction velocities and multiple destinations of efferent axons. Both the callosal projection and the projection to V-II were shown to originate primarily in layer II-III. However, approximately 10-20% of the callosal projection and 20-40% of the projection to V-II originated in layers IV and V. In contrast, the projection to the dorsal lateral geniculate nucleus originated nearly exclusively in layer VI, while corticotectal neurons occurred primarily in layer V. A significant number of corticotectal neurons were, however, found in layer IV. Thus, the above efferent systems were found to differ in their principal laminar origin and in the diffuseness of that origin. The origins of corticocortical projections were considerably more diffuse than those of corticofugal projections. In addition to differences in laminar origin, efferent systems also differed significantly in the conduction velocities of their axons. The projection to visual area II and to the lateral geniculate nucleus consisted primarily of very slowly conducting axons, while the projection to the superior colliculus was fast conduction. The callosal projection consisted of both slow and fact conduction axons. Finally, the question of branching of V-I efferent axons was addressed. Although the laminar origin of the projections to the contralateral hemisphere and to visual area II overlapped considerably, none of these corticocortical axons could be shown to project to both locations or to a subcortical destination. In contrast, approximately one-third of corticotectal axons were shown to project a collateral into the thalamus. Although the destination of this collateral is unclear, it is medial to the lateral geniculate nucleus and may be the pulvinar.

Origins of the projections of the superior colliculus to the dorsal lateral geniculate nucleus and the pulvinar in the rabbit.

The origins of the projections of the superior colliculus to the dorsal lateral geniculate nucleus and to the pulvinar in Dutch-belted rabbits were investigated using horseradish peroxidase (HRP) methods. Following injections of HRP in the dorsal lateral geniculate nucleus, retrogradely labeled neurons were found in the upper two-thirds of the stratum griseum superficiale of the ipsilateral superior colliculus. Most of the labeled somata were spindle-shaped, and their major axes tended to be perpendicular to the surface of the superior colliculus. In contrast, following injections of the pulvinar, labeled neurons were found in the lower third of the ipsilateral stratum griseum superficiale. In these cases, the labeled somata were larger than those labeled following dorsal lateral geniculate injections and were multipolar in shape.

Efferent projections of the septum in the Tegu lizard, Tupinambis nigropunctatus.

A H3 proline or H3 leucine mixture was injected into the septal region of the Tegu lizard in order to determine its efferent projections. The brains were processed according to standard autoradiographic technique and counterstained with cresyl violet. Septal projections were limited to either telencephalic or diencephalic areas. Intratelencephalic projections consisted of efferents to medial pallium, nucleus accumbens, bed nucleus of the anterior commissure, preoptic area and septum itself. Fibers entering the diencephalon projected to medial habenular nucleus, dorsomedial thalamic nucleus, dorsolateral thalamic area, periventricular nucleus of the hypothalamus, lateral hypothalamic area and mammillary nucleus. The results are discussed in relation to the efferent projections of the septum in other vertebrates.