Multiple Sources of Cholinergic Input to the Superior Olivary Complex.

The superior olivary complex (SOC) is a major computation center in the brainstem auditory system. Despite previous reports of high expression levels of cholinergic receptors in the SOC, few studies have addressed the functional role of acetylcholine in the region. The source of the cholinergic innervation is unknown for all but one of the nuclei of the SOC, limiting our understanding of cholinergic modulation. The medial nucleus of the trapezoid body, a key inhibitory link in monaural and binaural circuits, receives cholinergic input from other SOC nuclei and also from the pontomesencephalic tegmentum. Here, we investigate whether these same regions are sources of cholinergic input to other SOC nuclei. We also investigate whether individual cholinergic cells can send collateral projections bilaterally (i.e., into both SOCs), as has been shown at other levels of the subcortical auditory system. We injected retrograde tract tracers into the SOC in gerbils, then identified retrogradely-labeled cells that were also immunolabeled for choline acetyltransferase, a marker for cholinergic cells. We found that both the SOC and the pontomesencephalic tegmentum (PMT) send cholinergic projections into the SOC, and these projections appear to innervate all major SOC nuclei. We also observed a small cholinergic projection into the SOC from the lateral paragigantocellular nucleus of the reticular formation. These various sources likely serve different functions; e.g., the PMT has been associated with things such as arousal and sensory gating whereas the SOC may provide feedback more closely tuned to specific auditory stimuli. Further, individual cholinergic neurons in each of these regions can send branching projections into both SOCs. Such projections present an opportunity for cholinergic modulation to be coordinated across the auditory brainstem.

[Effect of the Na+, K(+)-ATPase modulation in neurons of the medulla oblongata on hemodynamic effects in spontaneously hypertensive rats].

The study was conducted in normotensive and spontaneously hypertensive rats anesthetized with urethane (1600 mg/kg of animal weight, intraperitoneally). It has been shown that in normotensive rats, injections of a specific inhibitor of Na+, K(+)-ATPase ouabain (10(-8)-10(-5) mol/l) in the populations of the neurons within nucleus of the solitary tract (NTS), paramedian reticular nucleus (PMn) and lateral reticular nucleus (LRN) were accompanied by the development of the hypertensive responses in a dose-dependent fashion. These data suggest that Na+, K(+)-ATPase of the neuron somatic membranes in the medullary cardiovascular nuclei is involved in neural control of the cardiovascular function, and its inhibition by microinjections of ouabain promotes the development of hypertension. In contrast to normotensive rats, ouabain injected in the medullary nuclei of spontaneously hypertensive animals induced either enhanced hypertensive or hypotensive responses. Biochemical analysis revealed that the activity of Na+, K(+)-ATPase in the microsomal fraction of the medulla oblongata of spontaneously hypertensive rats significantly exceeded its activity in the medulla oblongata of normotensive animals. Possible mechanisms of ouabain effects in spontaneously hypertensive rats have being discussed. Activation of Na+, K(+)-ATPase activity of the cardiovascular neurons with asparkam injections in the medullary nuclei resulted in hypotensive responses in both normotensive and spontaneously hypertensive rats.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Immunohistochemical study on the distribution of insulin-like growth factor-binding protein 4 in the central nervous system of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis.

In the present study, we used the SOD1(G93A) mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of insulin-like growth factor-binding protein 4 (IGFBP4) in the central nervous system. Decreased expression of IGFBP4 was obvious in the cerebral cortex, hippocampus, cerebellar cortex and inferior olive of SOD1(G93A) transgenic mice. In the cerebral cortex, there was a significant decrease in IGFBP4 immunoreactivity in the pyramidal cells. In the hippocampal formation, IGFBP4 immunoreactivity was also decreased in the pyramidal cells of CA1-3 areas and the granule cells of dentate gyrus. In the cerebellar cortex, IGFBP4 immunoreactivity was prominent in the granular layer in wtSOD1 transgenic mice, compared to that in SOD1(G93A) transgenic mice. IGFBP4 immunoreactivity was decreased in the inferior olive of SOD1(G93A) transgenic mice. This study, showing decreased IGFBP4 in different brain regions of SOD1(G93A) transgenic mice, may provide clues to understanding the differential susceptibility of neural structures in ALS, suggesting a role of IGFBP4 in an abnormality of cognitive and/or motor function in ALS. The mechanisms and functional implications of these decreases require elucidation.

Molecular, topographic, and functional organization of the cerebellar nuclei: analysis by three-dimensional mapping of the olivonuclear projection and aldolase C labeling.

The olivocerebellar climbing fiber projection pattern is closely correlated with the pattern of aldolase C expression in cerebellar Purkinje cells. Based on this expression pattern, the olivocerebellar projection can be classified into five "groups" of functional compartments. Each group originates from a subarea within the inferior olive that projects to multiple cortical stripes of Purkinje cells, all of which are either aldolase C positive or aldolase C negative. However, no equivalent compartmental organization has been demonstrated in the cerebellar nuclei (CN). Thus, in the CN of the rat, we systematically mapped the location of olivonuclear projections belonging to the five groups and determined their relationship to the expression of aldolase C in Purkinje cell axonal terminals. The CN were divided into caudoventral aldolase C-positive and rostrodorsal aldolase C-negative parts. The olivonuclear terminations from the five groups projected topographically to five separate compartments within the CN that partly crossed the traditional boundaries that define the fastigial, interposed, and dentate nuclei. Each compartment had mostly uniform cytoarchitecture and the same aldolase C expression (either positive or negative) that was found in the corresponding olivocortical projection. These results suggest a new view of the organization of the CN whereby the pattern of olivonuclear terminations links portions of different CN together. We propose that each compartment in the CN, along with its corresponding olivary subarea and cortical stripes, may be related to a different aspect of motor control.

Identification of aldolase C compartments in the mouse cerebellar cortex by olivocerebellar labeling.

Aldolase C (zebrin II) is expressed in Purkinje cells aligned in complicated longitudinal stripe-shaped compartments. The tight link between these aldolase C compartments and the topographic olivocerebellar projection to them has made it possible to identify each compartment as a target of a specific subarea of the inferior olive and thus as a functionally distinct entity in the rat. However, it is unknown whether the overall organization of aldolase C compartments is preserved in other mammals. In this study, we tried to clarify this organization in the mouse, which is more useful in genetic studies than the rat, by identifying each aldolase C compartment in terms of the olivocerebellar projection pattern. First, aldolase C compartments were reconstructed from serial sections throughout the cerebellar cortex. Aldolase C and olivocerebellar climbing fibers were then doubly labeled by small injections of biotinylated dextran amine into various areas of the inferior olive. Climbing fibers were topographically distributed on a specific linked pair of aldolase C compartments in the rostral and caudal cerebellum. The overall relationship between aldolase C compartments and the topographic olivocerebellar projection to them in the mouse was similar to that in the rat, except for some minor differences, suggesting that the aldolase C compartments and olivocerebellar projection are organized according to a common fundamental organization in the mouse and rat. This allowed the unequivocal identification of all aldolase C compartments in the mouse by referring to the definition and nomenclature in the rat.

Effects of cochlear ablation on choline acetyltransferase activity in the rat cochlear nucleus and superior olive.

Using microdissection and quantitative microassay, choline acetyltransferase (ChAT) activity was mapped in the cochlear nucleus (CN) and in the source nuclei of the olivocochlear bundle, the lateral superior olive and ventral nucleus of the trapezoid body. In control rats, gradients of ChAT activity were found within the major subdivisions of the CN and in the lateral superior olive. These gradients correlated with the known tonotopic organizations, with higher activities corresponding to locations representing higher sound frequencies. No gradient was found in the ventral nucleus of the trapezoid body. In rats surviving 7 days or 1 or 2 months after cochlear ablation, ChAT activity was increased 1 month after ablation in the anteroventral CN by 30-50% in most parts of the lesion-side and by 40% in the contralateral ventromedial part. ChAT activity in the lesion-side posteroventral CN was increased by approximately 40-50% at all survival times. Little change was found in the dorsal CN. Decreases of ChAT activity were also found ipsilaterally in the lateral superior olive and bilaterally in the ventral nucleus of the trapezoid body. Our results suggest that cholinergic neurons are involved in plasticity within the CN and superior olive following cochlear lesions.

The distribution and the modulation of tyrosine hydroxylase immunoreactivity in the lateral olivocochlear system of the guinea-pig.

It was previously shown that tyrosine hydroxylase (TH) immunoreactivity in the terminals of the lateral efferents of the cochlea is decreased by acoustic trauma and that sound preconditioning counteracted this decrease [Hear Res 174 (2002) 124]. Here we identify those neurons in the lateral olivocochlear system (LOC) in the brainstem that regulates the peripheral expression of TH in the cochlea. By employing retrograde tracing techniques, dextran-labeled neurons were found predominantly in the ipsilateral LOC system including lateral superior olive (LSO), and the surrounding periolivary regions (dorsal periolivary nucleus [DPO], dorsolateral periolivary nucleus [DLPO], lateral nucleus of trapezoid body [LNTB]). Employing immunocytochemistry, it was found that a control group had 35% of the ipsilateral LOC neurons positively stained with TH. Of the total population of TH neurons, 77% were double-stained (TH and dextran) in the LOC system. Acoustic trauma decreased the number of TH positive neurons in the LSO and the surrounding DLPO, and caused a reduction of TH fiber immunolabeling in these regions. Changes were not found in the DPO or the LNTB after acoustic trauma. Sound conditioning protected against the decrease of TH immunolabeling by acoustic trauma and increased the fiber staining for TH in the LSO and DLPO, but not in the DPO or the LNTB. These results provide evidence that TH positive neurons are present in the LOC system in the guinea-pig. It is now demonstrated that protection against acoustic trauma by sound conditioning has a central component that is governed by TH in the LSO and the surrounding periolivary DLPO region.

Dopaminergic olivocochlear neurons originate in the high frequency region of the lateral superior olive of guinea pigs.

Dopaminergic neurons are known to exist within the lateral superior olive (LSO). The LSO is the nucleus of origin of the lateral olivocochlear neurons, which project to the cochlea and synapse onto the primary afferents contacting the inner hair cells. We investigated whether the dopaminergic neurons in the LSO are part of the lateral olivocochlear neuron population. We combined intracochlear injections of a fluorescent retrograde tracer with immunofluorescent staining of tyrosine hydroxylase (TH). TH was used as a marker for dopaminergic neurons. After the injection with retrograde tracer most of the TH-labelled neurons in the LSO also contained the tracer, which directly demonstrates for the first time that the TH-labelled, dopaminergic neurons in the LSO are lateral olivocochlear neurons. TH-labelled neurons were not equally distributed over the LSO as is observed for the lateral olivocochlear neurons in general. TH-labelled neurons were almost exclusively seen in the medial, high frequency, limb of the LSO. Since the projection of the lateral olivocochlear neurons to the cochlea is known to be tonotopic, we investigated the TH-labelling in the cochlea as well. We found that the staining pattern of TH in the cochlea is in broad agreement with the distribution of TH-labelling in the LSO. Cochlear sections showed dense labelling in the basal and second, high frequency, turns and decreasing intensity of staining in the third turn, while the extreme apical, low frequency, turn was almost devoid of any positive TH-labelling. These observations imply that the dopaminergic neurons of the lateral olivocochlear system may play a role in the selective suppression of the high frequency fibers of the auditory system.

Postnatal changes in cytochrome oxidase expressions in brain stem nuclei of rats: implications for sensitive periods.

Previously, we reported that cytochrome oxidase (CO) activity in the rat pre-Bötzinger complex (PBC) exhibited a plateau on postnatal days (P) 3-4 and a prominent decrease on P12 (Liu and Wong-Riley, J Appl Physiol 92: 923-934, 2002). These changes were correlated with a concomitant reduction in the expression of glutamate and N-methyl-d-aspartate receptor subunit 1 and an increase in GABA, GABAB, glycine receptor, and glutamate receptor 2. To determine whether changes were limited to the PBC, the present study aimed at examining the expression of CO in a number of brain stem nuclei, with or without known respiratory functions from P0 to P21 in rats: the ventrolateral subnucleus of the solitary tract nucleus, nucleus ambiguus, hypoglossal nucleus, nucleus raphe obscurus, dorsal motor nucleus of the vagus nerve, medial accessory olivary nucleus, spinal nucleus of the trigeminal nerve, and medial vestibular nucleus (MVe). Results indicated that, in all of the brain stem nuclei examined, CO activity exhibited a general increase with age from P0 to P21, with MVe having the slowest rise. Notably, in all of the nuclei examined except for MVe, there was a plateau or decrease at P3-P4 and a prominent rise-fall-rise pattern at P11-P13, similar to that observed in the PBC. In addition, there was a fall-rise-fall pattern at P15-P17 in these nuclei, instead of a plateau pattern in the PBC. Our data suggest that the two postnatal periods with reduced CO activity, P3-P4 and especially P12, may represent common sensitive periods for most of the brain stem nuclei with known or suspected respiratory control functions.

Endogenous estrogen formation is neuroprotective in model of cerebellar ataxia.

The expression of aromatase, the enzyme that transforms testosterone into estradiol, was analyzed by reverse transcriptase polymerase chain reaction in the inferior olive of adult male rats. The expression of this messenger in the inferior olive suggests that this brain area may be able to synthesize estradiol. The neuroprotective role of estradiol in the inferior olive was then assessed in a model of cerebellar ataxia, achieved by the ip administration of 3-acetylpyridine (3-AP). In a first experiment, male Wistar rats were orchidectomized to diminish the plasmatic levels of testosterone, the direct precursor of estradiol. Immediately after castration, animals were implanted with a silicone tube that was either empty or filled with estradiol. One week later, animals were injected with 3-AP. Estradiol treatment resulted in a significant reduction in neuronal death in the olive. In a second experiment, animals were treated with fadrozole, an aromatase inhibitor, to assess the role of endogenous estradiol formation in neuroprotection. The results show that the inhibition of aromatase activity, and therefore the decrease in endogenous estrogen formation, increases the death in inferior olive. In conclusion, this study indicates that the inferior olive is a steroidogenic tissue and that olivary neurons are protected by exogenous and endogenous estradiol.

Conductive hearing loss results in changes in cytochrome oxidase activity in gerbil central auditory system.

Conductive hearing loss (CHL) restricts auditory input to an intact peripheral auditory system. Effects of deprivation on the central auditory system (CAS) have been debated, although a number of studies support the hypothesis that CHL can cause modification of CAS structure and function. The present study was designed to test the hypothesis that unilateral CHL results in a decrease in cytochrome oxidase (CO) activity in CAS nuclei that receive major afferent input from the affected ear. Gerbils at postnatal day 12 (P21) or 6-8 weeks underwent left unilateral CHL (malleus removal), cochlear ablation, or a sham surgical procedure. After a survival time of 48 hours or 3 weeks, animals were sacrificed and tissue was processed for cytochrome oxidase histochemistry. Optical density (OD) measurements were made from individual neurons in the anteroventral cochlear nucleus (AVCN) and from medial and lateral dendritic fields in the medial superior olivary nucleus (MSO), the lateral superior olivary nucleus, and the inferior colliculus. The width of the CO-stained neuropil in MSO was also measured as an estimate of dendritic length. OD measures were corrected to neutral areas of the brain. Cochlear ablation caused significant decreases in CO activity in left lower brainstem nuclei, particularly in adult animals. Following CHL, a significant decrease in CO activity was observed in the ipsilateral AVCN and a significant increase was observed in the contralateral AVCN. Cochlear ablation resulted in decreased width of MSO neuropil containing dendrites that receive primary input from the ablated ear. CHL resulted in a significant increase in the width of MSO neuropil on both sides of the brain in the P21 animals that survived 3 weeks but not in P21 animals that survived only 48 hours or in the adult animals. Unilateral CHL is associated with changes in CO activity in the AVCN and may affect MSO dendritic length in younger animals.

[Nitric oxide synthase in neurons of the vagal nucleus dorsalis and ganglion nodosum and its changes following acetylcholine inhalation in the norm and in experimental bronchial asthma]. / Nitrooksidsintaza neironov zadnego iadra i uzlovatogo gangliia bluzhdaiushchego nerva i ee izmeneniia pri ingaliatsiiakh atsetilkholina v norme i pri éksperimental'noi bronkhial'noi astme.

The expression NADPH-diaphorase and inducible NO-synthase (NOS) was studied in vagal nucleus dorsalis and ganglion nodosum neurons following acethylcholine inhalation in healthy rats and rats with ovalbumin-induced experimental bronchial asthma (BA). It was found that NOS activity regulation is mediated by cholinoreceptors; functioning of this mechanism is disturbed in hypoxic state. It is shown that both in conditions of physiological norm and in experimental BA, changes in NOS activity are determined by its constitutive, neuronal isoform.

Localization of Triton-insoluble cAMP-dependent kinase type RIbeta in rat and mouse brain.

In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RIalpha and beta, RIIalpha and beta). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RIbeta isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RIalpha and RIbeta isoforms is bound to the cytoskeleton. RIbeta labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RIalpha aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RIalpha isoforms, while the RIbeta isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RIalpha and beta isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions.

The human olivocochlear system: organization and development.

The goals of the present study were to identify olivocochlear neurons in the human brainstem, to establish the time course of their early development and to compare the organization of the human olivocochlear system to that of other mammals. To accomplish these goals, we used immunohistochemistry for choline acetyltransferase (ChAT) and calcitonin gene-related peptide (CGRP) in postmortem brainstems of human subjects ranging in age from 16 fetal weeks to 17 years. By immunostaining, we identified two classes of cells in the superior olivary complex: both classes were seen to be present from the twenty-first fetal week to the seventeenth year. Neurons which are immunostained only for ChAT are located primarily in the dorsomedial, ventral and ventrolateral sectors of the periolivary region. These neurons are predominantly bipolar or multipolar cells, and are probably homologous to medial olivocochlear neurons in other species. A second population of cells is immunoreactive for both ChAT and CGRP. This population includes a cluster of mostly small oval neurons, located on the dorsal edge of the olivary complex, and a variable number of cells found along the margin of the lateral olivary nucleus. These ChAT- and CGRP-immunoreactive cells are likely to be homologous to the lateral olivocochlear system in other mammals. With increasing age, the dorsal cluster of small cells shifts from its original cap-like position over the lateral olivary nucleus to become an extended column of cells lying among the fibers of the olivocochlear bundle.

Nitric oxide synthase in identified olivocochlear projection neurons in rat and guinea pig.

Nitric oxide (NO) is thought to be involved in the effects of amino acids at the level of cochlear hair cell afferents. Recently, the isoform of the NO-producing enzyme, neuronal NO synthase (nNOS), has been demonstrated in neuronal structures of the cochlea in rats and guinea pigs histochemically and immunohistochemically. To investigate the sources of cochlear NO, we injected Fluoro-Gold (FG) into the cochlea of rats and guinea pigs. Upon terminal uptake of the tracer and neuronal transport we observed FG in terminals at the base of inner (IHC) and outer hair cells (OHC) and in neurons of the spiral ganglion. Ganglion cells and terminals at the IHC were clearly nNOS-positive, while terminals at the OHC exhibited nNOS-immunoreactivity to a minor degree. The immunohistochemical investigation of the auditory brainstem showed that about one-fourth of the neurons of the superior olivary complex contained nNOS. The comparison with retrogradely labeled neurons showed that perikarya in the lateral superior olivary nucleus and, in particular, the medial nucleus of the trapezoid body were double-labeled. These results were similar in both, rat and guinea pig. Our data reveal that neurons of the superior olivary complex are likely to be additional sources of neuronal NOS in the cochlea.

Nitric oxide/cyclic guanosine monophosphate pathway in the peripheral and central auditory system of the rat.

The neuronal isoform of nitric oxide synthase (nNOS) and soluble guanylate cyclase (sGC) were localized in the cochlea, the cochlear nucleus (CN), and the superior olivary complex (SOC) of Fisher 344 rats. In the cochlea, nNOS was identified in spiral ganglion cells by using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and in situ hybridization. NADPH-diaphorase staining also was detected in blood vessels of the modiolus. By using immunohistochemistry against cyclic guanosine monophosphate, cochlear sGC activity was localized to pericytes in the spiral ligament as well as nerve fibers innervating outer hair cells. In the lower auditory brainstem, nNOS was localized to principal cells of the medial nucleus of the trapezoid body (MNTB) with NADPH-diaphorase histochemistry and in situ hybridization. NADPH-diaphorase activity also was observed in the lateral and medial superior olive (LSO and MSO, respectively), the superior periolivary nucleus (SPN), the ventral and lateral nuclei of the trapezoid body (VNTB and LNTB, respectively), and the ventral cochlear nucleus (VCN). Transcripts of the beta-subunit of sGC were localized in rat brainstem by using in situ hybridization. mRNA for sGC was expressed in neurons within the SPN, LSO, MSO, LNTB, MNTB, VNTB, and VCN. Highest levels of sGC expression were seen in the SPN. These results suggest that the NO/cGMP pathway is involved in both the ascending and descending pathways of the auditory brainstem.

Nitric oxide synthase in the auditory brain stem.

The auditory superior olivary complex (SOC), a group of inter-related brain stem nuclei, sends efferents to a variety of neuronal structures including the cochlea and the inferior colliculus. The present study conducted in rats and hamsters sought to investigate whether the neuroactive substance nitric oxide (NO) is produced in the rodent SOC. Immunohisto- and cytochemistry of the NO-synthesizing enzyme neuronal NO-synthase (nNOS), and histochemistry (NADPH-diaphorase activity of nNOS) was used. Perikarya stained by either method were found in the medial nucleus of the trapezoid body and in periolivary nuclei projecting to the cochlea, while terminals were observed mainly in the lateral superior olivary nucleus and in the superior paraolivary nucleus. These data demonstrate the existence of nNOS in the rodent SOC and provide evidence that nitric oxide acts as neuroactive substance in the auditory brain stem and the organ of Corti.

Development and differentiation of the anuran auditory brainstem during metamorphosis: an acetylcholinesterase histochemical study.

The time course of cell differentiation and the presence of histochemically defined areas in brainstem auditory nuclei were examined in developing bullfrogs, Rana catesbeiana, using cresyl violet staining and acetylcholinesterase (AChE) histochemistry. In the medulla, the dorsolateral nucleus (DLN) can be seen as a distinct structure in its adult location only at Gosner stage 40 and beyond. The majority of cells in the DLN are not fully differentiated until late metamorphic climax (stages 45-46) and early postmetamorphosis. The more ventral vestibular nucleus differentiates earlier (stage 37) than the DLN. Adult-like organization of auditory nuclei in the torus semicircularis (TS) of the midbrain cannot be reliably discerned until metamorphic climax stages. Cellular masses in the brainstem reveal AChE from the earliest stage examined (stage 27) but the intensity of staining differs among cell groups. Staining intensity in the DLN is at a peak in recently metamorphosed froglets. The time course of cell differentiation in the DLN precedes slightly or is coincident with the increased, transient presence of AChE. Staining of the superior olive stabilizes at a moderate level in early postmetamorphic stages. Ventral regions of the principal nucleus in the TS stain more intensely than dorsal regions beginning at stage 40. This dorsal-ventral gradient in staining persists in adult stages. There is a transient decline in staining of the laminar nucleus in metamorphic climax stages. Staining intensity in the magnocellular nucleus peaks during stages 40-46 and in early postmetamorphic froglets and then declines in adults, paralleling the pattern seen in the DLN. These data suggest that metamorphic climax and early froglet periods are an important developmental window for major differentiation and maturational events in the auditory brainstem.

Regional differences between the immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II alpha and beta isoforms in the brainstem of the rat.

The distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) alpha and beta isoforms in the brainstem of adult rats was investigated using an immunohistochemical method with two monoclonal antibodies which specifically recognize the alpha and beta isoform, respectively. We found that these isoforms were differentially expressed by neurons in the substantia nigra, red nucleus, dorsal cochlear nucleus, pontine nuclei and inferior olivary nucleus. Neurons in the inferior olivary nucleus express the alpha isoform, but not the beta isoform. In contrast, neurons in the substantia nigra, red nucleus and pontine nuclei were immunostained with the beta antibody, but not with the alpha antibody. In the dorsal cochlear nucleus, neurons in layers I and II were alpha-immunopositive, whereas neurons in layers III and IV were beta-immunopositive. Therefore, the distribution of the CaM kinase II alpha-immunopositive neurons is completely different from that of CaM kinase II beta-immunopositive neurons. Next we examined the possible coexistence of CaM kinase II alpha isoform and glutamate or that of CaM kinase II beta isoform and glutamic acid decarboxylase (GAD) in the single neuron by double immunofluorescence labelling using a pair of anti-alpha and anti-glutamate antibodies, or a pair of anti-beta and anti-GAD antibodies. The results indicated that neurons expressing anti-alpha immunoreactivity were also immunopositive against anti-glutamate antibody, and neurons expressing beta isoform were also immunopositive against anti-GAD antibody, suggesting that alpha-immunopositive neurons are classified as excitatory-type neurons, and on the contrary, beta-immunopositive neurons are classified as inhibitory-type neurons. In conclusion, the present study confirmed that alpha- and beta-isoforms of CaM kinase II are differentially expressed in the nuclei of the brainstem and have different roles.