Cholinergic boutons are closely associated with excitatory cells and four subtypes of inhibitory cells in the inferior colliculus.

Acetylcholine (ACh) is a neuromodulator that has been implicated in multiple roles across the brain, including the central auditory system, where it sets neuronal excitability and gain and affects plasticity. In the cerebral cortex, subtypes of GABAergic interneurons are modulated by ACh in a subtype-specific manner. Subtypes of GABAergic neurons have also begun to be described in the inferior colliculus (IC), a midbrain hub of the auditory system. Here, we used male and female mice (Mus musculus) that express fluorescent protein in cholinergic cells, axons, and boutons to look at the association between ACh and four subtypes of GABAergic IC cells that differ in their associations with extracellular markers, their soma sizes, and their distribution within the IC. We found that most IC cells, including excitatory and inhibitory cells, have cholinergic boutons closely associated with their somas and proximal dendrites. We also found that similar proportions of each of four subtypes of GABAergic cells are closely associated with cholinergic boutons. Whether the different types of GABAergic cells in the IC are differentially regulated remains unclear, as the response of cells to ACh is dependent on which types of ACh receptors are present. Additionally, this study confirms the presence of these four subtypes of GABAergic cells in the mouse IC, as they had previously been identified only in guinea pigs. These results suggest that cholinergic projections to the IC modulate auditory processing via direct effects on a multitude of inhibitory circuits.

Two classes of GABAergic neurons in the inferior colliculus.

The inferior colliculus (IC) is unique, having both glutamatergic and GABAergic projections ascending to the thalamus. Although subpopulations of GABAergic neurons in the IC have been proposed, criteria to distinguish them have been elusive and specific types have not been associated with specific neural circuits. Recently, the largest IC neurons were found to be recipients of somatic terminals containing vesicular glutamate transporter 2 (VGLUT2). Here, we show with electron microscopy that VGLUT2-positive (VGLUT2(+)) axonal terminals make axosomatic synapses on IC neurons. These terminals contain only VGLUT2 even though others in the IC have VGLUT1 or both VGLUT1 and 2. We demonstrate that there are two types of GABAergic neurons: larger neurons with VGLUT2(+) axosomatic endings and smaller neurons without such endings. Both types are present in all subdivisions of the IC, but larger GABAergic neurons with VGLUT2(+) axosomatic terminals are most prevalent in the central nucleus. The GABAergic tectothalamic neurons consist almost entirely of the larger cells surrounded by VGLUT2(+) axosomatic endings. Thus, two types of GABAergic neurons in the IC are defined by different synaptic organization and neuronal connections. Larger tectothalamic GABAergic neurons are covered with glutamatergic axosomatic synapses that could allow them to fire rapidly and overcome a slow membrane time constant; their axons may be the largest in the brachium of the IC. Thus, large GABAergic neurons could deliver IPSPs to the medial geniculate body before EPSPs from glutamatergic IC neurons firing simultaneously.

Characterization of the human central nucleus of the inferior colliculus.

The inferior colliculus (IC) is a major relay station for both ascending and descending auditory pathways. The IC is divided into three major regions, the external cortex (ECIC), the dorsal cortex (DCIC) and the central nucleus of the inferior colliculus (CNIC). While the ECIC and DCIC receive many non-auditory inputs, the CNIC receives predominantly auditory input ascending within the lateral lemniscus and descending input from the cerebral cortex. Recent work in animal models emphasizes the complexity of the CNIC and provides evidence for multiple ascending informational streams reaching this nucleus. Despite an abundance of research on the CNIC in laboratory animals, the microscopic anatomy and neurochemistry of the human CNIC is poorly understood. Herein, we utilize a combination of gross morphology, myelin staining, Nissl staining, histochemistry, immunohistochemistry and immunofluorescence to characterize the human CNIC. Our results indicate that the human CNIC occupies a volume of approximately 22.4 mm3 and includes over 420,000 neurons. The human CNIC is dominated by round/oval neurons arranged with their long axis parallel to fibrodendritic lamina. Additionally, the vast majority of CNIC neurons are associated with a perineuronal net, there is an abundance of tyrosine hydroxylase immunoreactive axons and puncta and neurons immunoreactive for glutamic acid decarboxylase. These results are largely consistent with observations in laboratory animals.

Morphological and neurochemical changes in GABAergic neurons of the aging human inferior colliculus.

It is well known that quality of hearing decreases with increasing age due to changes in the peripheral or central auditory pathway. Along with the decrease in the number of neurons the neurotransmitter profile is also affected in the various parts of the auditory system. Particularly, changes in the inhibitory neurons in the inferior colliculus (IC) are known to affect quality of hearing with aging. To date, there is no information about the status of the inhibitory neurotransmitter GABA in the human IC during aging. We have collected and processed inferior colliculi of persons aged 11-97 years at the time of death for morphometry and immunohistochemical expression of glutamic acid decarboxylase (GAD67) and parvalbumin. We used unbiased stereology to estimate the number of cresyl-violet and immunostained neurons. Quantitative real-time PCR was used to measure the relative expression of the GAD67 mRNA. We found that the number of total, GABAergic and PV-positive neurons significantly decreased with increasing age (p < 0.05). The proportion of GAD67-ir neurons to total number of neurons was also negatively associated with increasing age (p = 0.004), but there was no change observed in the proportion of PV-ir neurons relative to GABAergic neurons (p = 0.25). Further, the fold change in the levels of GAD67 mRNA was negatively correlated to age (p = 0.024). We conclude that the poorer quality of hearing with increasing age may be due to decreased expression of inhibitory neurotransmitters and the decline in the number of inhibitory neurons in the IC.

[Effects of sodium salicylate on the expressions of gamma-aminobutyricacid and glutamate and auditory response properties of the inferior colliculus neurons].

The effects of sodium salicylate (NaSA) on the expressions of gamma-aminobutyricacid (GABA) and glutamate (Glu), and auditory response properties of the inferior colliculus neurons in mice were studied. Thirty-six Kunming mice were divided into three groups: control group (saline injection); NaSA group (NaSA 450 mg/kg, i.p., each day for 15 d); NaSA + lidocaine group (NaSA 450 mg/kg + lidocaine 10 mg/kg, i.p., each day for 15 d). The expressions of GABA and Glu were examined with immunohistochemical method. The intensity-rate function, intensity-latency function and frequency-tuning curve were determined with extracellular electrophysiological recording. Results are as follows: (1) The expression of GABA in the NaSA and NaSA + lidocaine groups decreased remarkably compared with that in the control group; there was no noticeable difference between the NaSA and NaSA + lidocaine groups. The expression of Glu in the NaSA group increased significantly compared with that in the control and NaSA + lidocaine groups. No difference in the expression of Glu was found between the control and NaSA + lidocaine groups. (2) In NaSA group, the intensity-rate function displayed a non-monotonic pattern, rising at low intensity and descending at high intensity; the tip of frequency-tuning curves became broad after administration of NaSA. (3) The changes in intensity-rate function and intensity-latency function were not evident and the tips of the frequency-tuning curves sharpened in the NaSA + lidocaine group. These results suggest that administration of NaSA increases the expression of Glu-positive neurons and reduces that of GABA-positive neurons in the inferior colliculus. NaSA changes the auditory response properties of the inferior colliculus and lidocaine can reverse these changes.

Cytoarchitectural and functional abnormalities of the inferior colliculus in sudden unexplained perinatal death.

The inferior colliculus is a mesencephalic structure endowed with serotonergic fibers that plays an important role in the processing of acoustic information. The implication of the neuromodulator serotonin also in the aetiology of sudden unexplained fetal and infant death syndromes and the demonstration in these pathologies of developmental alterations of the superior olivary complex (SOC), a group of pontine nuclei likewise involved in hearing, prompted us to investigate whether the inferior colliculus may somehow contribute to the pathogenetic mechanism of unexplained perinatal death. Therefore, we performed in a wide set of fetuses and infants, aged from 33 gestational weeks to 7 postnatal months and died of both known and unknown cause, an in-depth anatomopathological analysis of the brainstem, particularly of the midbrain. Peculiar neuroanatomical and functional abnormalities of the inferior colliculus, such as hypoplasia/structural disarrangement and immunonegativity or poor positivity of serotonin, were exclusively found in sudden death victims, and not in controls. In addition, these alterations were frequently related to dysgenesis of connected structures, precisely the raphé nuclei and the superior olivary complex, and to nicotine absorption in pregnancy. We propose, on the basis of these results, the involvement of the inferior colliculus in more important functions than those related to hearing, as breathing and, more extensively, all the vital activities, and then in pathological conditions underlying a sudden death in vulnerable periods of the autonomic nervous system development, particularly associated to harmful risk factors as cigarette smoking.

Cloning, expression and pharmacology of the mouse 5-HT(4L) receptor.

Since most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT(4L) receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT(4L))expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT(4L) receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT(4S))and the long form (5-HT(4L)) of 5-HT4 receptors are expressed in all mouse and rat brain areas.

Fos-like immunoreactivity in central auditory neurons of the mouse.

Fos-like immunoreactivity was used to study sound-induced activation of neurons in the auditory brainstem. Immunoreactivity was assayed with a polyclonal antibody to Fos. In response to 6-kHz tone bursts, the pattern of staining was a band of immunoreactive neurons positioned at the tonotopically appropriate position within the cochlear nucleus and the inferior colliculus. The band was narrow at low sound pressure levels but wider along the tonotopic axis at higher sound levels. In response to noise bursts, the pattern was broader and often extended throughout the auditory nuclei. Often within this broad pattern were "sub-bands" of immunostained neurons, interspersed with bands of unstained neurons. With increasing sound pressure levels above 35-55 dB, the number of Fos-like immunoreactive neurons increased for the cochlear nucleus, superior olivary complex, and inferior colliculus. In the cochlear nucleus and inferior colliculus, the stained cells were small, and hence their activity would be difficult to sample in electrophysiological studies. In the medial nucleus of the trapezoid body, the stained neurons had larger somata and other characteristics of principal cells. Anesthesia with Nembutal or Avertin, but not with ketamine or urethane, decreased the number of Fos-like immunoreactive neurons in the cochlear nucleus. The different anesthetics produced more variable results in the inferior colliculus. In anesthetized, monaurally stimulated animals, the presence of staining in the contralateral cochlear nucleus indicates that some Fos-like immunoreactivity may be mediated by descending or commissural systems. These observations indicate that Fos assays are useful for studying the pattern of neuronal activation in the auditory system and may also be useful in studying the descending auditory pathways.

Fibroblast growth factor-2-like immunoreactivity in auditory brainstem nuclei of the developing and adult rat: correlation with onset and loss of hearing.

Fibroblast growth factor-2 (FGF-2; basic FGF) is widely distributed in the developing and adult brain and has numerous effects on cultured and lesioned neural cells. The physiological role of FGF-2 in the unlesioned nervous system, however, is still not understood. We have studied the distribution of FGF-2 in the developing, adult, and functionally impaired central auditory system of the rat using specific antibodies and peroxidase-antiperoxidase immunocytochemistry. FGF-2-like immunoreactivity (FGF-2-IR) occurred in neuronal cell bodies and/or nerve fibers but was very rarely observed in glial cells. Several auditory brainstem nuclei, including the superior paraolivary nucleus, the medial superior olive, the lateral and ventral trapezoid nuclei, and the central nucleus, as well as the external cortex of the inferior colliculus, were entirely devoid of FGF-2-IR. In the dorsal cochlear nucleus, the lateral superior olive, and the nuclei of the lateral lemniscus, FGF-2-IR was not detectable in nerve cell bodies prior to adult age. Neurons in the medial geniculate body exhibited FGF-2-IR only transiently, from postnatal day (P) 5 until P16. Neurons in the medial nucleus of the trapezoid body were immunoreactive from P8 onwards. FGF-2-IR in anteroventral and posteroventral cochlear neurons disappeared at P14, i.e., at the onset of hearing, but immunoreactivity returned after P21. A transient expression of FGF-2 around the time when hearing function commences was observed in the dorsal cortex of the inferior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)

Parvalbumin, calbindin D-28k, and calretinin immunoreactivity in the ascending auditory pathway of horseshoe bats.

In the subcortical auditory system of Rhinolophus rouxi, antibodies directed against the calcium-binding proteins parvalbumin, calbindin D-28k, and calretinin yield partly overlapping and partly complementary labeling patterns which are described in detail for each nucleus. The most general features of the labeling patterns are that: 1) Parvalbumin is a potent marker for large and heterogeneous populations of cells and puncta (presumed axon terminals) throughout the auditory pathway. 2) Immunostaining with the monoclonal calbindin-antiserum was typically absent or sparse in most auditory brainstem centers, but prominent in auditory nerve fibers and in cells of the medial geniculate body (MGB). 3) Calretinin label is abundant but more restricted to subsets of auditory nuclei or subpopulations of cells than parvalbumin. 4) Calcium-binding proteins are useful markers to define particular subregions or cell types in auditory nuclei: for example, i) different labeling patterns are obtained within the nuclei of the lateral lemniscus and adjacent tegmental zones; ii) in the inferior colliculus both calbindin- and calretinin-antisera yield similar regional specific staining patterns, but label different cell types; iii) subregions of the medial geniculate body have characteristic profiles of calcium-binding proteins; and iv) analyses of different nuclei showed that there is no simple common denominator for cells characterized by the expression of particular calcium-binding proteins, nor does labeling correspond in a straightforward way with specific functional systems. 5) there are profound differences between the calbindin labeling patterns seen in Rhinolophus and those in other mammals.

Serotonergic innervation of the auditory brainstem of the Mexican free-tailed bat, Tadarida brasiliensis.

Anatomical and electrophysiological evidence suggests that serotonin alters the processing of sound in the auditory brainstem of many mammalian species. The Mexican free-tailed bat is a hearing specialist, like other microchiropteran bats. At the same time, many aspects of its auditory brainstem are similar to those in other mammals. This dichotomy raises an interesting question regarding the serotonergic innervation of the bat auditory brainstem: Is the serotonergic input to the auditory brainstem similar in bats and other mammals, or are there specializations in the serotonergic innervation of bats that may be related to their exceptional hearing capabilities? To address this question, we immunocytochemically labeled serotonergic fibers in the brainstem of the Mexican free-tailed bat, Tadarida brasiliensis. We found many similarities in the pattern of serotonergic innervation of the auditory brainstem in Tadarida compared with other mammals, but we also found two striking differences. Similarities to staining patterns in other mammals included a higher density of serotonergic fibers in the dorsal cochlear nucleus and in granule cell regions than in the ventral cochlear nucleus, a high density of fibers in some periolivary nuclei of the superior olive, and a higher density of fibers in peripheral regions of the inferior colliculus compared with its core. The two novel features of serotonergic innervation in Tadarida were a high density of fibers in the fusiform layer of the dorsal cochlear nucleus relative to surrounding layers and a relatively high density of serotonergic fibers in the low-frequency regions of the lateral and medial superior olive.

Distribution of calcium-binding protein calbindin-D28k in the auditory system of adult and developing rats.

Calbindin-D28k (CaBP) is a calcium-binding protein, which appears to be involved in the buffering of free intracellular calcium and may thereby contribute to calcium homeostasis. This study attempted to determine the distribution pattern of CaBP immunoreactivity in the central auditory system of adult rats and during development, when calcium ions play key roles in several aspects of nerve cell function. It was found that most steps during CaBP development occur postnatally in the central auditory system. With the exception of the lateral superior olive, the ventral and the intermediate nuclei of the lateral lemniscus, and the auditory cortex, which already express CaBP prenatally, CaBP immunoreactivity is not present before postnatal day 2 (P2). Development proceeds until about P24, when the pattern characteristic of adult animals can be seen. There was no detectable sequence in CaBP development from lower to higher stations in the auditory pathway, i.e., the different nuclei appear to express CaBP independently of each other, indicating that intrinsic, rather than peripheral, maturation processes may predominantly influence CaBP expression. Neurons in four brainstem nuclei (the lateral superior olive, the ventral and intermediate nuclei of the lateral lemniscus, and the central nucleus of the inferior colliculus) express CaBP only transiently. In these nuclei, CaBP immunoreactivity peaks between P6 and P18, which coincides with the period of synapse stabilization. Therefore, CaBP may play a specific role during neuronal development, by buffering the concentration of intracellular free Ca2+, which may be necessary for modification of synaptic efficiency.

Distribution of GABAA, GABAB, and glycine receptors in the central auditory system of the big brown bat, Eptesicus fuscus.

Quantitative autoradiographic techniques were used to compare the distribution of GABAA, GABAB, and glycine receptors in the subcortical auditory pathway of the big brown bat, Eptesicus fuscus. For GABAA receptors, the ligand used was 35S-t-butylbicyclophosphorothionate (TBPS) for GABAB receptors, 3H-GABA was used as a ligand in the presence of isoguvacine to block binding to GABAA sites; for glycine, the ligand used was 3H-strychnine. In the subcortical auditory nuclei there appears to be at least a partial complementarity in the distribution of GABAA receptors labeled with 35S-TBPS and glycine receptors labeled with 3H-strychnine, GABAA receptors were concentrated mainly in the inferior colliculus (IC) and medial geniculate nucleus, whereas glycine receptors were concentrated mainly in nuclei below the level of the IC. Within the IC, there was a graded spatial distribution of 35S-TBPS binding; the most dense labeling was in the dorsomedial region, but very sparse labeling was observed in the ventrolateral region. There was also a graded spatial distribution of 3H-strychnine binding. The most dense labeling was in the ventral and lateral regions and the weakest labeling was in the dorsomedial region. Thus, in the IC, the distribution of 35S-TBPS was complementary to that of 3H-strychnine. GABAB receptors were distributed at a low level throughout the subcortical auditory nuclei, but were most prominent in the dorsomedial part of the IC.

Cochlear electrical stimulation: influence of age of implantation on Fos immunocytochemical reactions in inferior colliculi and dorsal cochlear nuclei of the rat.

The influence of age at the time of implantation of a stimulating electrode unilaterally in the inner ear on central auditory pathways was investigated in rats deafened shortly after birth. Immunoreactivity for Fos served as a functional marker of neuronal activity. Electrodes were implanted in the left cochlea of rats aged 3 weeks or 4 months. Stimulation lasted 45 minutes, then rats were sacrificed and tissues processed for immunocytochemistry. The younger animals showed significantly more neurons with Fos immunoreactivity bilaterally in the dorsal cochlear nuclei (DCN) and inferior colliculi (IC) than the older rats or control animals with normal hearing receiving the same stimulation. Activity was more prominent in the left DCN and right IC. The results show that electrical stimulation of the inner ear is more effective in younger animals in eliciting gene expression associated with development of a functional network in the auditory pathways. This suggests that deaf children should be provided with cochlear implants as early as possible.

GABA and glycine in the central auditory system of the mustache bat: structural substrates for inhibitory neuronal organization.

The distribution and morphology of neurons and axonal endings (puncta) immunostained with antibodies to gamma-aminobutyric acid (GABA) and glycine (Gly) were analyzed in auditory brainstem, thalamic, and cortical centers in the mustache bat. The goals of the study were (1) to compare and contrast the location of GABAergic and glycinergic neurons and puncta, (2) to determine whether nuclei containing immunoreactive neurons likewise have a similar concentration of puncta, (3) to assess the uniformity of immunostaining within a nucleus and to consider regional differences that were related to or independent of cytoarchitecture, and (4) to compare the patterns recognized in this bat with those in other mammals. There are nine major conclusions. (1) Glycinergic immunostaining is most pronounced in the hindbrain. (2) In the forebrain, GABA alone is present. (3) Some nuclei have GABAergic or glycinergic neurons exclusively; a few have neither. (4) Although there is sometimes a close relationship between the relative number of immunopositive neurons and the density of the puncta, just as often there is no particular correlation between them; this reflects the fact that many GABAergic and glycinergic neurons project beyond their nucleus of origin. (5) Even nuclei devoid of or with few GABAergic or glycinergic neurons contain relatively abundant numbers of puncta; some neurons receive axosomatic terminals of each type. (6) In a few nuclei there are physiological subregions with specific local patterns of immunostaining. (7) The patterns of immunostaining resemble those in other mammals; the principal exceptions are in nuclei that, in the bat, are hypertrophied (such as those of the lateral lemniscus) and in the medial geniculate body. (8) Cellular colocalization of GABA and Gly is specific to only a few nuclei. (9) GABA and glutamic acid decarboxylase (GAD) immunostaining have virtually identical distributions in each nucleus. Several implications follow. First, the arrangements of GABA and Gly in the central auditory system represent all possible patterns, ranging from mutually exclusive to overlapping within a nucleus to convergence of both types of synaptic endings on single neurons. Second, although both transmitters are present in the hindbrain, glycine appears to be dominant, and it is often associated with circuitry in which precise temporal control of aspects of neuronal discharge is critical. Third, the auditory system, especially at or below the level of the midbrain, contains significant numbers of GABAergic or glycinergic projection neurons. The latter feature distinguishes it from the central visual and somatic sensory pathways.

Development of calretinin immunoreactivity in the brainstem auditory nuclei of the barn owl (Tyto alba).

The early development of calretinin immunoreactivity (CR-IR) was described in the auditory nuclei of the brainstem of the barn owl. CR-IR was first observed in the auditory hindbrain at embryonic day (E17) and a day later (E18) in the inferior colliculus. In each of the auditory nuclei studied, CR-IR did not develop homogeneously, but began in the regions that map high best frequencies in the adult barn owl. In the hindbrain, CR-IR was first observed in the rostromedial regions of the cochlear nucleus magnocellularis and the nucleus laminaris, and in the dorsal regions of the nucleus angularis and in the nucleus of the lateral lemniscus. In the inferior colliculus, CR-IR began in the ventral region of the central core. The edge of these gradients moved along the future tonotopic axes during the development of all nuclei studied, until adult patterns of CR-IR were achieved about a week after hatching.

GAD- and GABA-immunoreactivity in the ascending auditory pathway of horseshoe and mustached bats.

A comparative study of the immunostain to antibodies directed against glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the ascending auditory pathway was carried out in horseshoe bats (Rhinolophus rouxi) and mustached bats (Pteronotus parnellii). In both species GAD/GABA-positive puncta (presumed axonal boutons) and GAD/GABA-positive cells were found in the cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus the inferior colliculus, and the medial geniculate body. General features of the immunostaining pattern in the auditory pathway agree with observations in other mammals. Quantitative analysis of puncta distribution shows that many auditory centers are characterized by subregional differences in puncta density and distribution. This indicates local differences in putatively inhibitory input related to connectivity and tonotopic organization. The following species characteristic features were found: 1) The dorsal non-laminated portion of the dorsal cochlear nucleus in horseshoe bats lacks the GAD/GABA-immunoreactive cells typical for the ventral laminated portion and the dorsal cochlear nucleus of other species. Clearly, a cytoarchitectonic specialization is accompanied by a loss of putatively GABAergic local inhibitory circuits. 2) The ventral division of the medial geniculate body of the mustached bat lacks GAD/GABA-immunopositive cells. Such cells are present in the horseshoe bat and other mammals. This finding implies functional differences in the organization of the medial geniculate body within the same mammalian order.

Localization of two high-threshold potassium channel subunits in the rat central auditory system.

The firing pattern of auditory neurons is determined in part by the type of voltage-sensitive potassium channels expressed. The expression patterns for two high-threshold potassium channels, Kv3.1 and Kv3.3, that differ in inactivation properties were examined in the rat auditory system. The positive activation voltage and rapid deactivation kinetics of these channels provide rapid repolarization of action potentials with little effect on action potential threshold. In situ hybridization experiments showed that Kv3.3 mRNA was highly expressed in most auditory neurons in the rat brainstem, whereas Kv3.1 was expressed in a more limited population of auditory neurons. Notably, Kv3.1 mRNA was not expressed in neurons of the medial and lateral superior olive and a subpopulation of neurons in the ventral nucleus of the lateral lemniscus. These results suggest that Kv3.3 channels may be the dominant Kv3 subfamily member expressed in brainstem auditory neurons and that, in some auditory neurons, Kv3.1 and Kv3.3 may coassemble to form functional channels. The localization of Kv3.1 protein was examined immunohistochemically. The distribution of stained somata and neuropil varied across auditory nuclei and correlated with the distribution of Kv3.1 mRNA-expressing neurons and their terminal arborizations, respectively. The intensity of Kv3.1 immunoreactivity varied across the tonotopic map in the medial nucleus of the trapezoid body with neurons responding best to high-frequency tones most intensely labeled. Thus, auditory neurons may vary the types and amount of K(+) channel expression in response to synaptic input to subtly tune their firing properties.

Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain.

A novel anti-beta(3) subunit-specific GABA(A) receptor (GABA(A)R) antibody has been prepared by immunizing a rabbit with a bacterial fusion protein of the large intracellular loop of the beta(3) subunit. The antiserum immunoprecipitated the solubilized GABA(A) receptor. The anti-beta(3) antibody was affinity purified on immobilized beta(3) large intracellular loop peptide. In immunoblots, the purified antibody reacted with a 57 KDa peptide. Immunocytochemistry with the affinity-purified antibody has revealed the localization of the beta(3) subunit in the rat brain. A comparative study with the immunocytochemical distribution of the beta(2) subunit has also been performed. There are areas of the brain and cell types where the distribution of beta(2) and beta(3) overlap (i.e., cerebral cortex, cerebellum,and most layers of the olfactory bulb). There are also clear differences in the expression of beta(3) and beta(2) in other brain areas and cell types. Thus, high beta(3) but low or no beta(2) expression was observed in the corpus striatum and in granule cells of the olfactory bulb. In the hippocampus the expression of beta(3) was considerably higher than that of beta(2), but some hippocampal interneurons showed high expression of beta(2). High beta(2) but little or no expression of beta(3) was observed in thalamic nuclei, substantia nigra, globus pallidus, inferior colliculus and the short axon cells of the olfactory bulb.

Common patterns of increased and decreased fos expression in midbrain and pons evoked by noxious deep somatic and noxious visceral manipulations in the rat.

Immunohistochemical detection of the protein product (Fos) of the c-fos immediate early gene was used to study neuronal activation in the rostral pons and midbrain of halothane-anesthetised rats following noxious deep somatic or noxious visceral stimulation. In animals exposed only to halothane anesthesia, Fos-like immunoreactive (IR) neurons were located in the midbrain periaqueductal gray matter, tectum, and parabrachial nucleus. Following noxious stimulation of hindlimb muscle, knee joint, vagal cardiopulmonary, or peritoneal nociceptors, there was, compared to halothane-only animals, a significant increase in the numbers of Fos-like (IR) cells in the caudal ventrolateral periaqueductal gray and the intermediate gray lamina of the superior colliculus. Given the general agreement that increased Fos expression is a consequence of increased neuronal activity, the finding that a range of noxious deep somatic and noxious visceral stimuli evoked increased neuronal activity in a discrete, caudal ventrolateral periaqueductal gray region is consistent with previous suggestions that this region is an integrator of deep noxious evoked reactions. The noxious deep somatic and noxious visceral manipulations also evoked, compared to halothane-only animals, reductions in the numbers of Fos-like IR cells in the stratum opticum of the superior colliculus and the unlaminated portion of the external subnucleus of the inferior colliculus. To our knowledge this is the first report of reductions in Fos-expression in the tectum evoked by noxious stimulation. In separate experiments, the effects of noxious deep somatic and noxious visceral manipulations on arterial pressure and heart rate were measured. The noxious visceral manipulations evoked substantial and sustained falls in arterial pressure (15-45 mmHg), and heart rate (75-100 bpm), whereas the depressor and bradycardiac effects of the noxious deep somatic manipulations were weaker, not as sustained, or entirely absent. As similar distributions and numbers of both increased and decreased Fos-like IR cells were observed after each of the deep noxious manipulations, it follows that the deep noxious evoked increases and decreases in Fos expression were not secondary to the evoked depressor or bradycardiac effects.