Function of the sexually dimorphic ear of the American bullfrog, Rana catesbeiana: brief review and new insight.

The dimorphic ear of the bullfrog, Rana catesbeiana, has long been enigmatic. The male's tympanic membrane (TM) area approximates twice the area of the female's; however, similar size differences in the area of the columellar footplate were not observed between the sexes. Hence, the male's hearing is expected to be more sensitive than the female's but this is not the case. Asking what offsets the advantage of the large TM, we applied a series of experiments to the auditory system. Male and female audiograms based on stimulation with airborne sound and on both multi-unit responses from the brain and alternating cochlear potentials ('microphonics') showed equal sensitivity and a small difference in frequency response; at low frequencies the male was more sensitive than the female. Amputating the columella and stimulating the stump with mechanical vibration showed that for an equal microphonic response, the male's footplate vibrated with lower amplitude than the female's footplate. Mechanically stimulating the TM of the intact ear replicated this result, excluding the involvement of the mechanical lever. The TM of the male weighs five times the TM of the female, and artificial loading of the TM of either sex greatly reduced the ear's sensitivity. Hence, the male's excessive area ratio (TM to columellar footplate) is offset by the heavier cartilage cushion on the male's TM, damping the TM's response to sound. This is corroborated by experimentally artificially loading the TM. The product of area ratio and footplate vibration amplitude would result in similar stimulation of the inner ear in the two sexes.

Immunohistological characterization of striatal and amygdalar structures in the telencephalon of the fire-bellied toad Bombina orientalis.

The subpallium of the fire-bellied toad Bombina orientalis was studied by means of enzyme-histological detection of NADPH-diaphorase and immunohistological demonstration of aspartate, GABA, calretinin, choline-acetyl transferase, Leu-and Met-enkephalin, neuropeptide Y, 5-hydroxy-tryptamine (serotonin), somatostatin, substance P and tyrosine-hydroxylase. As in other vertebrates, the striato-pallidum is characterized by GABA-, substance P- and enkephalin-immunoreactivity. Neurons and fibers differing in immunoreactivity are arranged in layers. Choline-acetyl transferase-immunoreactive neurons were found in a position corresponding to the mammalian cholinergic cell-group (Ch4-group), which therefore may be homologous to the nucleus basalis of Meynert. Within the amygdaloid complex, the cortical and lateral (vomeronasal) nuclei are similar in calretinin-, GABA-, NADPH-diaphorase-, enkephalin, substance P- and neuropeptide Y-(immuno)histology. The medial and central amygdaloid nuclei reveal a dense peptidergic innervation, and the medial amygdala additionally exhibits serotonergic fibers and cell bodies staining for neuropeptides and tyrosine-hydroxylase. Differences between Bombina and other anuran species exist, such as the absence of cholinergic neurons in the striatum. Our findings corroborate the view based on recent studies on the hodology and cytoarchitecture of the anuran telencephalon that the anuran ventral telencephalon contains most of the structures found in the mammalian brain. This concerns a septal region, a dorsal and ventral striato-pallidum including a nucleus accumbens and an amygdaloid complex consisting of a central, cortical and vomeronasal amygdala. The only major difference appears to concern the lack of a basolateral amygdala.

Chemoarchitecture of the anuran auditory midbrain.

The anuran torus semicircularis consists of several subnuclei that are part of the ascending auditory pathway as well as audiomotor interface structures. Additionally, recent anatomical studies suggest that the midbrain tegmentum is an integral part of the audiomotor network. To describe the chemoarchitecture of these nuclei, taking into account the toral subdivisions, we investigated the distribution of serotonin, leucine-enkephalin, substance P, tyrosine-hydroxylase, dopamine D2-receptor, parvalbumin, aspartate, GABA, and estrogen-binding protein-immunoreactivity in the midbrain of Bombina orientalis, Discoglossus pictus and Xenopus laevis. In the torus semicircularis, the highest density of immunoreactive fibers and terminals for all transmitters was found in the laminar nucleus. Parvalbumin-like immunoreactivity was highest in the principal nucleus, and D2-receptor-like immunoreactivity was uniformly distributed throughout the torus. In the tegmentum, axons and/or dendrites were stained with all antibodies except estrogen-binding protein. Additionally, heavily stained enkephalin and substance P-immunopositive fiber plexus were found in the lateral and dorsal tegmentum. The immunostainings revealed no qualitative differences between the three species. Immunopositive cell bodies were labeled in several brain areas, the connectivity of which with torus and tegmentum is discussed on the background of functional questions. The putative neuromodulatory innervation of both the laminar nucleus of the torus semicircularis and the tegmentum may be the anatomical basis for the influence of the animal's endogenous state on the behavioral reaction to sensory stimuli. These data corroborate earlier anatomical and physiological findings that the neurons of these nuclei are key elements in the audio-motor interface.

[Effect of monaural deprivation on maturation of the auditory pathway in the Mongolian gerbil (Meriones unguiculatus)]. / Einfluss monauraler Deprivation auf die Reifung der Hörbahn bei der mongolischen Wüstenrennmaus (Meriones unguiculatus).

Several investigations prove that monaural acoustic deprivation induces detrimental changes in the auditory pathway. So far the major interest focused on degenerative processes on the deprived side, while effects on the contralateral untreated side were not taken into consideration. This ontogenetic study investigates effects of early monaural acoustic deprivation on the functional development of the acoustically evoked auditory brainstem response (ABR) in the Mongolian gerbil. Young gerbils were deafened by a single intracochlear application of neomycin sulfate. The ABR development on the contralateral side differed in a late developmental period from that of normally hearing animals. These changes persisted until adulthood. Between day 55 and day 90 a significant 5 dB decrease of the ABR thresholds and a significant shortening of the interpeak latency II-V occurred. The results confirm that a complete monaural deprivation during the sensible or critical period leads on the contralateral side to plastic or compensatory changes in the auditory brainstem.

Integration of ascending and descending inputs in the auditory midbrain of anurans.

In anurans, the midbrain torus semicircularis is involved in auditory processing and audio-motor integration. In this study, we examined the influence of descending forebrain projections on the auditory response properties and hence the audiomotor transmission of mesencephalic interface neurons. In order to investigate response integration, we performed intracellular recordings from torus neurons in an isolated brain preparation of Discoglossus pictus and Bombina orientalis and stimulated the auditory nerve, striatum, and the dorsal thalamus electrically with single pulses. Stimulation of all three sites could evoke responses in torus neurons that were either excitatory, inhibitory, or a mixture of both, with durations of up to several hundred milliseconds. Further, striatum and thalamus were activated by pulse trains (10-20 Hz, 50 pulses) immediately before stimulating the auditory nerve with single pulses. Thus, responses of torus neurons to "auditory" input were facilitated or suppressed for up to 2 min by striatum stimulation or only suppressed by thalamus stimulation. Intracellular labeling of recorded neurons revealed that response modulation by descending input mostly occurred in laminar nucleus neurons. These results suggest that descending forebrain projections to mesencephalic audiomotor interface neurons may play an important role in modifying acoustically guided behavior in anurans.

Significance of forebrain structures in acoustically guided behavior in anurans.

In order to test the hypothesis that the forebrain is involved in controlling acoustically guided behaviour, we carried out behavioural studies in combination with brain lesions, neuroanatomical and electrophysiological experiments in males and females of different species of frogs. Whereas the dorsomedial pallium plays no or only a minor role, the striatum, the septum, and the preoptic area potentially influence the behaviour because they send parallel descending projections to different premotor and motor networks in the brainstem. These parallel projections may be the basis for the variability seen in the behaviour.

Influence of descending forebrain projections on processing of acoustic signals and audiomotor integration in the anuran midbrain.

We tested the role of descending projections for auditory processing and audiomotor integration in the anuran torus semicircularis. Intracellular recordings were made from isolated brain preparations, impaled neurons were stained. Auditory neurons responded to electrical stimulation of striatum and/or dorsal thalamus, they integrated forebrain and auditory nerve inputs. High frequency stimulation in striatum or thalamus changed the auditory response of torus neurons located in the laminar subnucleus. Our results suggest that the laminar nucleus is the primary target of forebrain projections, which provides a basis for modulation of acoustically guided behaviour.

In vitro and in vivo responses of saccular and caudal nucleus neurons in the grassfrog (Rana temporaria).

We present results from in vitro and in vivo studies of response properties of neurons in the saccular and caudal nuclei in the frog. In the in vitro studies the saccular nerve of the isolated brain was stimulated with electrical pulses. In the in vivo experiments, the neurons were stimulated by dorso-ventral vibrations of the intact animal. We identified six response types: (1) primary-like cells with short latencies and follow repetition rates up to 100 Hz; (2) phasic cells responding only to the first pulse in a train; (3) bursting cells firing several spikes in response to any stimulation; (4) late responders with very long latencies; (5) integrator cells showing facilitated responses, and (6) inhibitory cells inhibited by saccular nerve stimulation. The cells have comparable sensitivity and frequency characteristics to the primary fibres (BF 10-80 Hz, thresholds from 0.01 cm/s2) and enable a sophisticated analysis of vibrational stimuli.

Effect of Structural Variation within Lipophilic Lariat Ether Phosphonic Acid Monoesters on the Selectivity and Efficiency of Competitive Alkali Metal Cation Extraction into Chloroform.

Lipophilic lariat ether phosphonic acid monoethyl esters with systematic crown ether ring size variation from 12-crown-4 to 24-crown-8 are utilized for competitive alkali metal cation extractions from aqueous solutions into chloroform. Effective alkali metal cation extraction from weakly acidic, neutral, and basic aqueous solutions is achieved. With 4, 5, and 6 oxygens in the crown ether rings, selectivities for Li(+), Na(+), and K(+), respectively, are observed. An 18-crown-6 phosphonic acid monoester exhibits excellent extraction selectivity for K(+) with K(+)/Li(+) and K(+)/Na(+) > 100. The lipophilic group attachment site, as well as the crown ether ring size, is shown to influence the extraction selectivity for the lariat ether phosphonic monoesters.

Morphology and axonal projection patterns of auditory neurons in the midbrain of the painted frog, Discoglossus pictus.

Acoustic signals are extensively used for guiding various behaviors in frogs such as vocalization and phonotaxis. While numerous studies have investigated the anatomy and physiology of the auditory system, our knowledge of intrinsic properties and connectivity of individual auditory neurons remains poor. Moreover, the neural basis of audiomotor integration still has to be elucidated. We determined basic response patterns, dendritic arborization and axonal projection patterns of auditory midbrain units with intracellular recording and staining techniques in an isolated brain preparation. The subnuclei of the torus semicircularis subserve different tasks. The principal nucleus, the main target of the ascending auditory input, has mostly intrinsic neurons, i.e., their dendrites and axons are restricted to the torus itself. In contrast, neurons of the magnocellular and the laminar nucleus project to various auditory and non-auditory processing centers. The projection targets include thalamus, tegmentum, periaqueductal gray, medulla oblongata, and in the case of laminar neurons--the spinal cord. Additionally, tegmental cells receive direct auditory input and project to various targets, including the spinal cord. Our data imply that both auditory and premotor functions are implemented in individual toral and tegmental neurons. Their axons constitute parallel descending pathways to several effector systems and might be part of the neural substrate for differential audiomotor integration.

A method for the induction of a cochlea-specific auditory deprivation in the gerbil (Meriones unguiculatus).

The neurophysiological effects of early electrical stimulation on the development and neural plasticity of the central auditory system in prelingually deafened children with cochlear implants are still unknown. Many of these basic questions can be answered systematically only in animal experiments. Meriones unguiculatus is a well-established animal model in hearing research. Deafening is produced by a single intracochlear application of an ototoxic aminoglycoside antibiotic (neomycin sulfate) on the 14th day after birth (DAB), i.e. before the late natural onset of hearing on the 16th DAB. A single application of the antibiotic abolishes auditory brainstem responses (ABR) to clicks completely and reduces sensitivity to low frequency tonebursts by 50 dB SPL. Scanning electron microscopy results show a destruction of the stereocilia of the inner and outer hair cells of the basal and medial cochlear turn and a reduction of those in the apical turn. Our method avoids a systemic application of antibiotics and can be used in studies dealing with the consequences of different forms of auditory deprivation, neuronal compensation processes or with ontogenetic studies and chronic electrostimulation in an animal model.

The use of in vitro preparations of the isolated amphibian central nervous system in neuroanatomy and electrophysiology.

In the present study an isolated preparation of the complete anuran central nervous system (CNS) is described which can be kept alive for several days and allows tracing, immunohistochemical and electrophysiological studies. A simple perfusion chamber is being used in which the isolated CNS preparation is superfused with oxygenated Ringer. The use of an isolated CNS has many advantages including: (1) virtually all areas are easily accessible at the same time without having the problem of blood vessels that hinder access; (2) large lesions and massive tracer applications are possible without survival problems of the animal, and tracers will not be translocated by blood circulation; (3) since pulsations caused by the pressure changes of blood circulation do not occur, intracellular recordings are comparatively easy and stable; and (4) this approach offers the possibility of working on the same brain for several days by storing the preparation in a refrigerator overnight at low temperatures, thus allowing extensive utilization of a single preparation and reduction in the number of experimental animals required. Some applications to the anuran auditory system illustrate that the isolated anuran CNS is well-suited for a variety of neuroanatomical and physiological techniques.

Audio-motor interface in anurans.

Like males of many anuran species, fire-bellied toads (Bombina orientalis) call antiphonally, which demonstrates an auditory input into the call-generating network. Males produce their calls by an inspiratory airstream, which is generated exclusively by contraction of the muscles of the buccal cavity. The painted frog (Discoglossus pictus) possesses a combined inspiratory and expiratory call mechanism, and also uses only buccal muscles. These muscles are controlled by branchial motoneurons, which receive vocal premotor input mainly from the pretrigeminal nucleus. The interconnections between the auditory pathway and the vocal pathway were examined by neuroanatomical tracing and intracellular recording. Mesencephalic auditory nuclei, laminar and magnocellular nucleus of the torus semicircularis, and tegmental nuclei constitute strong descending efferents, which, in turn, form collaterals that terminate in vocal premotor nuclei. These findings imply fast audio-vocal interfacing, which is a prerequisite for the control of antiphonal calling.

A comparative phylogenetic study of the distribution of cerebellar GABAA/benzodiazepine receptors using radioligands and monoclonal antibodies.

In mammalian cerebella the distribution of high affinity GABAA and benzodiazepine binding sites does not parallel each other. Differences in the molecular structure of the receptor complex or conformational changes have been proposed to explain the lack of a co-localization of these binding sites. Using radio- and immunohistochemistry we were able to obtain similar results for cerebella of non-mammalian species, indicating that the respective distribution of the binding sites is well conserved throughout vertebrate evolution. The gamma-aminobutyric acid agonist [3H]muscimol and the beta-subunit specific antibody bd-17 strongly labeled the granular layer. The radioligands for the benzodiazepine binding site [3H]flunitrazepam and [3H]Ro 15-1788 revealed species variation in the ratio of benzodiazepine receptor density in the granular and molecular layers. In human and pig cerebellum, the localization of antigenic sites recognized by the alpha-subunit specific antibody bd-24 deviates from the distribution of the benzodiazepine binding sites.

A comparative study of auditory sensitivity in the genus Bufo (amphibia).

Multi-unit response from the torus semicircularis were recorded to obtain neural audiograms for five different species, subspecies and races of the genus Bufo:Bufo b.bufo, Bufo b. spinosus, two races of Bufo viridis, and Bufo americanus. All of the audiograms were clearly bimodal, with a low frequency peak and a high frequency peak. A less pronounced but distinguishable intermediate peak seems to be an indication of trimodality. In the low-frequency range, the audiograms of the five groups differed little or not at all. There were pronounced differences, however, in the high-frequency range. This frequency band includes the frequencies of the conspecific mating calls. The best matching between call frequency and auditory best frequency was found in the Bufo viridis group. While it is likely that factors other than the signal characteristics in the vocal repertoire may shape the sensitivity of the auditory system, it is notable that those anuran species in which the mating call clearly facilitates reproduction have a more pronounced sensitivity peak at the frequencies in their mating calls.

Sensitivity, range and temperature dependence of hearing in the grass frog and fire-bellied toad.

Multi-unit recordings from the torus semicircularis of the fire-bellied toad (Bombina bombina L.) and the grass frog (Rana t. temporaria L.) were used to obtain threshold vs. frequency curves for these anurans. The effect of body temperature on these audiograms was tested over a range of 10-28° C for the toad and 5-20° C for the frog. The range of frequencies audible to the fire-bellied toad at a body temperature of 21° C extends to 2400-3000 Hz. Threshold is relatively low in three regions: 300-450 Hz, 700-900 Hz and 1200-1700 Hz. The auditory system is most sensitive in the low frequency region. The audiograms of both species depend greatly on temperature. As temperature is increased sensitivity is enhanced, particularly at low and intermediate frequencies. Grass frogs are maximally sensitive at temperatures as low as 15° C, whereas the auditory threshold of fire-bellied toads continue to fall as the temperature is raised from 16° C to 22° C. Hearing evidently is adapted to different temperature ranges in the two species, and these correspond to the temperatures at which the animals engage in mating behavior.