Stefano O. Casalotti

Gap junctions in the inner ear: Comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals

The distribution and size of gap junctions (GJ) in the sensory epithelia of the inner ear have been examined in a reptile (gecko), birds (chicken and owl), and mammals (mouse, guinea pig, gerbil, and bat), and the connexin composition of GJs in the mammalian inner ear has been assessed. Freeze fracture revealed a common pattern of GJ distribution in auditory and vestibular sensory epithelia in the different vertebrate classes. In all these tissues, GJs are numerous, often occupying more than 25% of the plasma membrane area of supporting cells and sometimes composed of more than 100,000 channels. Screening for 12 members of the connexin family in the mammalian inner ear by RT‐PCR, Western blotting, and immunohistochemistry revealed four connexin isotypes, cx26, cx30, cx31, and cx43, in the cochlea and three, cx26, cx30, and cx43, in the vestibular organs. With antibodies characterised for their specificity, cx26 and cx30 colocalised in supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type 1 fibrocytes of the spiral ligament. No other connexin was detected in these regions. Cx31 was localised among type 2 fibrocytes below the spiral prominence, a region where cx30 was not expressed and cx26 expression appeared to be low. Cx43 was detected only in the region of “tension fibrocytes” lining the inner aspect of the otic capsule. This suggests separate functional compartments in the cochlea. In addition to cx26 and cx30, cx43 was detected in supporting cells of the vestibular sensory epithelia. Where cx26 and cx30 were colocalised, double immunogold labelling of thin sections showed both cx26 and cx30 evenly distributed in individual GJ plaques, a pattern consistent with the presence of heteromeric connexons. Coimmunoprecipitation of cochlear membrane proteins solubilised with a procedure that preserves the oligomeric structure of connexons confirmed the presence of heteromeric cx26/cx30 connexons. Heteromeric cx26/cx30 connexons may be unique to the inner ear, which could be one factor underlying the non‐syndromic character of the deafness caused by mutations in cx26. J. Comp. Neurol. 467:207–231, 2003.

The Inner Ear Contains Heteromeric Channels Composed of Cx26 and Cx30 and Deafness-Related Mutations in Cx26 Have a Dominant Negative Effect on Cx30

Cx26 and cx30 co-localize in tissues of the mammalian cochlea. Transfected HeLa cells were used to examine interactions between cx26 and cx30 and the effects on cx30 of four point mutations in cx26 that are associated with dominantly inherited hearing loss—W44S, G59A, D66H and R75W. When co-expressed, wtcx26 and wtcx30 trafficked to the same gap junction plaques. Cells transferred neurobiotin but not Lucifer Yellow, which passes freely through cx26 channels, suggesting cx30 affects the properties of cx26. G59A and D66H had a perinuclear localization when expressed alone but trafficked to the membrane when co-expressed with cx30. Co-expression of W44S, G59A or R75W with cx30, significantly reduced neurobiotin transfer in comparison with cells expressing cx30 only. These results indicate that cx26 and cx30 can oligomerize to form heteromeric connexons and demonstrate a dominant negative effect of some cx26 mutants on cx30. Immunogold labeling of thin sections of the cochlea showed both cx26 and cx30 distributed evenly on both sides of individual gap junction profiles. Immunoprecipitation of cochlear membrane proteins, isolated by procedures that preserve connexons, with either cx30 or cx26 antibodies precipitated both cx26 and cx30. Following co-injection of Lucifer Yellow and neurobiotin into individual supporting cells of the organ of Corti in cochlear slices, neurobiotin transferred to many cells, but Lucifer Yellow was retained in the injected cell. These observations are consistent with junctions composed of cx26/cx30 heteromeric connexons in the cochlea. The functional disruption caused by some cx26 mutations upon such heteromeric channels may underlie the non-syndromic nature of their effects on hearing.

Postnatal Touch Stimulation Acutely Alters Corticosterone Levels and Glucocorticoid Receptor Gene Expression in the Neonatal Rat

Environmental manipulation early in life can alter the development of the hypothalamic-pituitary-adrenal (HPA) axis by mechanisms that are still unclear. The aim of the present work was to study the acute effects of postnatal touch stimulation, in an attempt to understand the mechanism by which touch stimulation early in life alters the HPA response to stress in adult animals. Rat pups were gently brushed for 15 min daily during the 1st postnatal week. Serum corticosterone levels were determined by radioimmunoassays, while glucocorticoid receptor (GR) gene expression was assayed by semiquantitative reverse transcriptase-polymerase chain reaction. Touch stimulation induced a significant decrease (30–36%) in serum corticosteroid secretion during the 1st and 2nd postnatal day as compared to the unstimulated group. In contrast, GR gene expression in the touch stimulation group was significantly increased in several brain areas such as the hippocampus (19–21%), frontal cortex (26–34%) and midbrain (15–24%). The results thus indicate that neonatal touch stimulation causes acute hormone- secretion and gene-expression changes within the period of stimulation. These changes may be the underlying cause for the permanent changes that have been observed in adult animals touch-stimulated as neonates.

Connexins and Gap Junctions in the Inner Ear

Mutations in the genes for three different isotypes of the gap junction channel protein connexin are associated with deafness. This indicates an important role for gap junctions in auditory function and provides an opportunity to explore structure-function relationships in the connexin molecule. We have been examining the distribution of gap junctions and the pattern of connexin expression in the mature inner ear and during development, and the effect of specific mutations on the processing and functionality of the expressed connexin proteins in an in vitro system.

The presence of opioid receptors in rat inner ear

Opioid peptides have been identified in the inner ear but relatively little information is available about the expression and distribution of their receptors. The aim of the present study was therefore to identify and localize the mu (MOR), delta (DOR) and kappa (KOR) opioid receptor subtypes within the rat cochlea. The expression of these opioid receptor subtypes was determined by reverse transcriptase-polymerase chain reaction followed by nested polymerase chain reaction analysis. Amplification of RNAs from rat cerebral cortex (positive control) and rat cochlea with MOR, DOR and KOR primers resulted in products of the predicted lengths, 564, 356 and 276 bp, respectively. Restriction digestion confirmed the identity of these products. All three receptor subtypes were identified in the cochlea and further characterized by immunocytochemistry. DOR and KOR immunoreactivity was found in inner and outer hair cells, bipolar cells of the spiral ganglion and interdental cells of the limbus. In contrast, no MOR immunoreactivity was observed in the inner and outer hair cells, and interdental cells. All three types of receptor fibers were also detected in the bipolar cells and nerve fibers within the spiral ganglion. In addition, MOR- and KOR-containing nerve fibers were observed in the limbus. These findings are the first report of the presence of all three classical opioid receptors in the inner ear and suggest that these receptors may have both presynaptic and postsynaptic roles.

Opioid modulation of GABA release in the rat inferior colliculus.

BackgroundThe inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication.ResultsUsing a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that μ rather than δ or κ opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for μ opiate receptors and relatively few neurons co-stained for both proteins.ConclusionThe results suggest that μ-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.

Morphine induces short-lived changes in G-protein gene expression in rat prefrontal cortex

We have utilized a reverse transcriptase-polymerase chain reaction (RT-PCR) methodology followed by enzymatic restriction analysis to detect changes in G-protein mRNA levels in morphine-treated rats. The relative distribution of mRNA levels for Galpha(o) Galpha(i1), Galpha(i2), Gbeta(1) and Gbeta(2) in the nucleus accumbens, striatum, locus coeruleus and prefrontal cortex was found to be similar to that previously estimated with other techniques. Morphine-induced changes of G-protein mRNA levels were detected only in the prefrontal cortex. Acute treatments (30 mg/kg, intraperitoneally) resulted in a significant increase of Galpha(o) mRNA and significant decreases of Galpha(i1) and Galpha(i2) mRNAs. Chronic morphine administration (10-50 mg/kg over 14 days, intraperitoneally) increased Gbeta(1) and Galpha(i1) and Galpha(i2) mRNAs levels to 148%, 410% and 451% of control, respectively. G-protein mRNA returned to control levels within 48 h of termination of the chronic treatments. The morphine-induced changes in G-protein mRNA levels may reflect changes in gene expression and could result in changes in G-protein levels affecting signal transduction pathways in chronically treated animals.

The existence of opioid receptors in the cochlea of guinea pigs

Several independent investigations have demonstrated the presence of opioid peptides in the inner ear organ of Corti and in particular in the efferent nerve fibers innervating the cochlear hair cells. However, the precise innervation pattern of opioid fibers remains to be investigated. In the present study the expression of opioid receptors and their peptides is demonstrated in young adult guinea pig cochlea. Opioid receptors are mainly expressed in hair cells of the organ of Corti and in inner and outer spiral bundles with different characteristics for each type of receptor. Co‐localization studies were employed to compare the distribution of mu‐, delta‐ and kappa‐opioid receptors and their respective peptides, β‐endorphin, leu‐enkephalin and dynorphin. Additionally, immunostaining of synaptophysin was used in this study to identify the presynaptic site. Immunoreactivity for enkephalin and dynorphin was found in the organ of Corti. Leu‐enkephalin was co‐localized with synaptophysin prominently in the inner spiral bundle (ISB). Dynorphin was co‐localized with synaptophysin in both inner and outer spiral bundles. Delta‐opioid receptor was most prominently co‐localized with its peptide in the ISB bundle. Kappa‐opioid receptor was seemingly present with dynorphin in both inner and outer spiral bundles. The co‐staining of both peptides and receptors with synaptophysin in the same areas suggests that some of the opioid receptors may act as auto‐receptors. The results provide further evidence that opioids may function as neurotransmitters or neuromodulators in the cochlea establishing the basis for further electrophysiological and pharmacological investigations to understand better the roles of the opioid system in auditory function.

Stress, anxiety and peripheral benzodiazepine receptor mRNA levels in human lymphocytes

Peripheral benzodiazepine receptor (PBR) mRNA levels were measured in lymphocytes obtained from a cohort of university students and clinically diagnosed anxious patients. The average level of PBR mRNA was decreased in anxious patients compared to a control group. This data confirms previously published results, but it also indicates that PBR mRNA levels cannot be used as a sole diagnostic measure of anxiety because the range of the individual PBR mRNA levels of the anxious group overlapped the range of the PBR mRNA levels of the control group. PBR mRNA levels in students following academic examinations were increased in some individuals and decreased in others. In the same cohort of students individual levels of cortisol and prolactin were predominantly increased and decreased respectively. There was no correlation between the individual changes in the hormone levels or PBR mRNA, which suggests that each of these parameters is affected by different environmental and physiological factors. Lymphocyte PBR mRNA measurement is a useful additional methodology for studying human stress responses however, its use in clinical studies would require the elucidation of PBRs physiological role.

The opioid receptors in inner ear of different stages of postnatal rats

There is increasing evidence that the opioid system has a role in hearing. To provide further evidence for such a role, the expression of opioid receptor mRNAs and proteins in the inner ear of rats was studied during development from birth (P0) to postnatal day 16 (P16). A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect changes in the expression of delta- (DOR) kappa- (KOR) and mu- (MOR) opioid receptor mRNAs in rat cochleae at P0, P4, P8 and P16. Expression of DOR mRNA levels steadily increased from P0 to P8 with no further increases by P16. KOR mRNA was expressed at a relatively high level at P0 and P4 followed by a decrease while MOR mRNA was expressed at a low level at P0 and P4 followed by an increase by P8 and P16. Immunocytochemical labelling of inner ear sections revealed unique developmental and distribution patterns of opioid receptors. In the organ of Corti DOR immunoreactivity (DOR-IR) was detected in hair cells from P4. In contrast MOR-IR was present only in supporting cells at P0-P16. In the spiral ganglion all three receptor subtypes were expressed from P0 on nerve cell soma and qualitatively appeared to increase with age. Also DOR-IR and MOR-IR were detected at P8 and P16 in nerve fibers within the spiral ganglion. In the limbus DOR-IR was detected at P8 and P16 on cells proximal to the tectorial membrane while MOR-IR was detected more distally. In general these findings demonstrate that within the inner ear each receptor subtype follows specific temporal and spatial developmental patterns, some of which may be associated to the onset of hearing. The data provide further evidence that the opioid system may play a role in the development and functioning of the inner ear.