Larry F. Hughes

Inhibitory Neurotransmission, Plasticity and Aging in the Mammalian Central Auditory System

SUMMARY Aging and acoustic trauma may result in partial peripheral deafferentation in the central auditory pathway of the mammalian brain. In accord with homeostatic plasticity, loss of sensory input results in a change in pre- and postsynaptic GABAergic and glycinergic inhibitory neurotransmission. As seen in development, age-related changes may be activity dependent. Age-related presynaptic changes in the cochlear nucleus include reduced glycine levels, while in the auditory midbrain and cortex, GABA synthesis and release are altered. Presumably, in response to age-related decreases in presynaptic release of inhibitory neurotransmitters, there are age-related postsynaptic subunit changes in the composition of the glycine (GlyR) and GABAA (GABAAR) receptors. Age-related changes in the subunit makeup of inhibitory pentameric receptor constructs result in altered pharmacological and physiological responses consistent with a net down-regulation of functional inhibition. Age-related functional changes associated with glycine neurotransmission in dorsal cochlear nucleus (DCN) include altered intensity and temporal coding by DCN projection neurons. Loss of synaptic inhibition in the superior olivary complex (SOC) and the inferior colliculus (IC) likely affect the ability of aged animals to localize sounds in their natural environment. Age-related postsynaptic GABAAR changes in IC and primary auditory cortex (A1) involve changes in the subunit makeup of GABAARs. In turn, these changes cause age-related changes in the pharmacology and response properties of neurons in IC and A1 circuits, which collectively may affect temporal processing and response reliability. Findings of age-related inhibitory changes within mammalian auditory circuits are similar to age and deafferentation plasticity changes observed in other sensory systems. Although few studies have examined sensory aging in the wild, these age-related changes would likely compromise an animals ability to avoid predation or to be a successful predator in their natural environment.

Gap detection deficits in rats with tinnitus: a potential novel screening tool.

The study describes a novel method for tinnitus screening in rats by use of gap detection reflex procedures. The authors hypothesized that if a background acoustic signal was qualitatively similar to the rats tinnitus, poorer detection of a silent gap in the background would be expected. Rats with prior evidence of tinnitus at 10 kHz (n = 14) exhibited significantly worse gap detection than controls (n = 13) when the gap was embedded in a background similar to their tinnitus. No differences between tinnitus and control rats were found with 16 kHz or broadband noise backgrounds, which helped to rule out explanations related to hearing loss or general performance deficits. The results suggest that gap detection reflex procedures might be effective for rapid tinnitus screening in rats.

d-Methionine provides excellent protection from cisplatin ototoxicity in the rat

Cisplatin (CDDP) is a widely used chemotherapeutic agent. Unfortunately, CDDP is highly ototoxic. We tested D-methionine (D-Met), a sulfur containing compound, as an otoprotectant in male Wistar rats. Complete data sets were obtained for five groups of five animals each, including a treated control group (16 mg/kg CDDP), an untreated control group (administered an equivalent volume of saline) and three groups that received either 75, 150, or 300 mg/kg D-Met 30 min prior to the 16 mg/kg CDDP dosing. Auditory brainstem response (ABR) thresholds were obtained in response to clicks, and 1 kHz, 4 kHz, 8 kHz, and 14 kHz toneburst stimuli, before and 3 days after drug administration. Scanning electron microscopy (SEM) was used to examine the outer hair cells of the apical, middle and basal turns of the cochlea. Animal weight was measured on the first and final day. D-Met provided excellent otoprotection even at the lowest level with complete otoprotection obtained for the 300 mg/kg dosing as measured by both ABR and SEM. D-Met also markedly reduced weight loss and mortality. All animals receiving D-Met (15/15) survived to the end of the study period as opposed to only 5/10 of the treated controls.

Prevention of noise- and drug-induced hearing loss with d-methionine ☆

A number of otoprotective agents are currently being investigated. Various types of agents have been found in animal studies to protect against hearing loss induced by cisplatin, carboplatin, aminoglycosides, or noise exposure. For over a decade we have been investigating D-methionine (D-met) as an otoprotective agent. Studies in our laboratory and others around the world have documented D-mets otoprotective action, in a variety of species, against a variety of ototoxic insults including cisplatin-, carboplatin-, aminoglycoside- and noise-induced auditory threshold elevations and cochlear hair cell loss. For cisplatin-induced ototoxicity, protection of the stria vascularis has also been documented. Further D-met has an excellent safety profile. D-met may act as both a direct and indirect antioxidant. In this report, we provide the results of three experiments, expanding findings in D-met protection in three of our translational research areas: protection from platinum based chemotherapy-, aminoglycoside- and noise-induced hearing loss. These experiments demonstrate oral D-met protection against cisplatin-induced ototoxicity, D-met protection against amikacin-induced ototoxicity, and D-met rescue from permanent noise-induced hearing loss when D-met is initiated 1h after noise exposure. These studies demonstrate some of the animal experiments needed as steps to translate a protective agent from bench to bedside.

Age-related changes in GABAA receptor subunit composition and function in rat auditory system

A decline in the ability to discriminate speech from noise due to age-related hearing loss (presbycusis) may reflect impaired auditory information processing within the central nervous system. Presbycusis may result, in part, from functional loss of the inhibitory neurotransmitter GABA. The present study assessed age-related changes of the GABA(A) receptor in the inferior colliculus of young-adult, middle-aged, and aged rats related to: (i) receptor subunit composition and (ii) receptor function. Western blotting was used to measure protein levels of selected GABA(A) receptor subunits in preparations obtained from the inferior colliculus of Fischer 344 and Fischer 344/Brown-Norway F1 hybrid rats. In both strains, the aged group exhibited significant increases in gamma1 subunit protein and a decrease in alpha1 subunit protein. To examine the functional consequence of this putative age-related subunit change, we measured the ability of exogenous GABA to flux/translocate chloride ions into microsac preparations derived from Fischer 344 inferior colliculus. GABA-mediated chloride influx was significantly increased in samples prepared from the inferior colliculus of aged animals. Together with previous studies, these results strongly suggest an age-related change in GABA(A) receptor composition. These changes may reflect a compensatory up-regulation of inhibitory function in the face of significant loss of presynaptic GABA release. These findings provide one example of plastic neurotransmitter receptor changes which can occur during the ageing process.

Dorsal cochlear nucleus responses to somatosensory stimulation are enhanced after noise‐induced hearing loss

Multisensory neurons in the dorsal cochlear nucleus (DCN) achieve their bimodal response properties [Shore (2005) Eur. J. Neurosci., 21, 3334–3348] by integrating auditory input via VIIIth nerve fibers with somatosensory input via the axons of cochlear nucleus granule cells [Shore et al. (2000) J. Comp. Neurol., 419, 271–285; Zhou & Shore (2004)J. Neurosci. Res., 78, 901–907]. A unique feature of multisensory neurons is their propensity for receiving cross‐modal compensation following sensory deprivation. Thus, we investigated the possibility that reduction of VIIIth nerve input to the cochlear nucleus results in trigeminal system compensation for the loss of auditory inputs. Responses of DCN neurons to trigeminal and bimodal (trigeminal plus acoustic) stimulation were compared in normal and noise‐damaged guinea pigs. The guinea pigs with noise‐induced hearing loss had significantly lower thresholds, shorter latencies and durations, and increased amplitudes of response to trigeminal stimulation than normal animals. Noise‐damaged animals also showed a greater proportion of inhibitory and a smaller proportion of excitatory responses compared with normal. The number of cells exhibiting bimodal integration, as well as the degree of integration, was enhanced after noise damage. In accordance with the greater proportion of inhibitory responses, bimodal integration was entirely suppressive in the noise‐damaged animals with no indication of the bimodal enhancement observed in a sub‐set of normal DCN neurons. These results suggest that projections from the trigeminal system to the cochlear nucleus are increased and/or redistributed after hearing loss. Furthermore, the finding that only neurons activated by trigeminal stimulation showed increased spontaneous rates after cochlear damage suggests that somatosensory neurons may play a role in the pathogenesis of tinnitus.

Age-Related Changes in the Inhibitory Response Properties of Dorsal Cochlear Nucleus Output Neurons: Role of Inhibitory Inputs

Age-related hearing loss frequently results in a loss in the ability to discriminate speech signals, especially in noise. This is attributable, in part, to a loss in temporal resolving power and ability to adjust dynamic range. Circuits in the adult dorsal cochlear nucleus (DCN) have been shown to preserve signal in background noise. Fusiform cells, major DCN output neurons, receive focused glycinergic inputs from tonotopically aligned vertical cells that also project to the ventral cochlear nucleus. Glycine-mediated inhibition onto fusiform cells results in decreased tone-evoked activity as intensity is increased at frequencies adjacent to characteristic frequency (CF). DCN output is thus shaped by glycinergic inhibition, which can be readily assessed in recordings from fusiform cells. Previous DCN studies suggest an age-related loss of markers for glycinergic neurotransmission. The present study postulated that response properties of aged fusiform cells would show a loss of inhibition, resembling conditions observed with glycine receptor blockade. The functional impact of aging was examined by comparing response properties from units meeting fusiform-cell criteria in young and aged rats. Fusiform cells in aged animals displayed significantly higher maximum discharge rates to CF tones than those recorded from young-adult animals. Fusiform cells of aged rats displayed significantly fewer nonmonotonic CF rate-level functions and an age-related change in temporal response properties. These findings are consistent with an age-related loss of glycinergic input, likely from vertical cells, and with findings from other sensory aging studies suggesting a selective age-related decrement in inhibitory amino acid neurotransmitter function.

Age-related loss of the GABA synthetic enzyme glutamic acid decarboxylase in rat primary auditory cortex

Age-related changes within the auditory brainstem typically include alterations in inhibitory neurotransmission and coding mediated by GABA and glycinergic circuits. As part of an effort to evaluate the impact of aging on neurotransmission in the higher auditory centers, the present study examined age-related changes in the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in rat primary auditory cortex (AI), which contains a vast network of intrinsic and extrinsic GABAergic circuits throughout its layers. Message levels of the two GAD isoforms found in brain, GAD(65) and GAD(67), and GAD(67) protein levels were compared in young adult, middle-aged and aged rats using in situ hybridization and quantitative immunocytochemistry, respectively. For comparison, age-related GAD changes were also assessed in the parietal cortex and hippocampus. Significant age-related decreases in GAD(65&67) messages were observed in AI layers II-VI of aged rats relative to their young adult cohorts. The largest changes were identified in layer II (GAD(65): -26.6% and GAD(67): -40.1%). GAD(67) protein expression decreased significantly in parallel with mRNA decreases in all layers of AI. Adjacent regions of parietal cortex showed no significant GAD(67) protein changes among the age groups, except in layer IV. As previously described, GAD(67) message and protein levels in selected hippocampal regions were significantly reduced in aged rats. Age-related GAD reductions likely reflect decreases in both metabolic and pre-synaptic GABA levels suggesting a plastic down-regulation of normal adult inhibitory GABA neurotransmission. Consistent with the present findings, functional studies in primate visual cortex and preliminary studies in AI find coding changes suggestive of altered inhibitory processing in aged animals. An age-related loss of normal adult GABA neurotransmission in AI would likely alter temporal coding properties and could contribute to the loss in speech understanding observed in the elderly.

D-Methionine protects against cisplatin damage to the stria vascularis.

D-Methionine (D-met) protects against cisplatin (CDDP)-induced hearing loss and outer hair cell loss (Campbell et al., 1996). However, D-mets protective effects on the stria vascularis has not been previously investigated. The purpose of this study was to examine, using semi-quantitative analysis, whether D-met also protects the stria vascularis. We removed a basal turn section of the stria vascularis from five groups of five male Wistar rats each: (1) a CDDP-treated control group receiving a 30 min i.p. infusion of 16 mg/kg CDDP, (2) a saline-injected control group receiving an equivalent volume of saline, and (3) three groups injected with either 75, 150, or 300 mg/kg D-methionine (D-met) i.p. 30 min prior to receiving the 16 mg/kg CDDP dosing. Using transmission electron microscopy and light microscopy, we analyzed strial volume (i.e. edema), marginal cell damage classification (bulging and/or compression), and relative optical density (ROD) ratios (i.e. depletion of marginal cell cytoplasmic organelles). All three levels of D-met provided complete protection against marginal cell bulging and/or compression but only partial protection against strial edema. At 300 mg/kg, D-met significantly reduced ROD ratio degradation in the spiral prominence and middle stria vascularis regions. In Reissners membrane region, values from the D-met pretreated group were not significantly different from either the treated or untreated control groups suggesting only partial protection for that area. Protection of marginal cell cytoplasmic organelles was also noted. In summary, D-met partially or fully protects the stria vascularis from several types of CDDP-induced damage.

GABAergic inputs shape responses to amplitude modulated stimuli in the inferior colliculus

The inferior colliculus (IC) is an important auditory processing center receiving inputs from lower brainstem nuclei, higher auditory and nonauditory structures, and contralateral IC. The IC, along with other auditory structures, is involved in coding information about the envelope of complex signals. Biologically relevant acoustic signals, including animal vocalizations and speech, are spectrally and temporally complex and display amplitude and frequency variations over time. Certain IC neurons respond selectively over a narrow range of modulation frequencies to sinusoidally amplitude modulated (SAM) stimuli. Responses to SAM stimuli can be measured in terms of discharge rate, with rate plotted against the modulation frequency to generate rate modulation transfer functions (rMTF). A role for the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), in shaping selective responses to SAM stimuli has been suggested. The present study examined the role of GABA in shaping responses to SAM stimuli in the IC of anesthetized chinchilla. Responses from 94 IC neurons were obtained before, during and after iontophoretic application of the GABA(A) receptor antagonist bicuculline methiodide. Complete responses to SAM stimuli were obtained from 55 extensively tested neurons, displaying band-pass (38) and low-pass rMTFs (17). For neurons showing band-pass rMTFs, GABA(A) receptor blockade selectively increased discharge rate at low modulation frequencies for 14 units, increased discharge near the best modulation frequency for 12 units. For neurons showing low-pass rMTFs, GABA(A) receptor blockade selectively increased discharge rate at low modulation frequencies for nine units. GABA(A) receptor blockade consistently reduced peak modulation gain, producing low-pass gain functions in a subset of IC neurons. In support of previous findings suggesting that selective temporal responses to SAM stimuli are coded in lower brainstem nuclei, temporal responses to SAM stimuli were relatively unaffected by GABA(A) receptor blockade. These findings support a role for GABA in shaping selective rate responses to SAM stimuli for a subset of chinchilla IC neurons.