Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.

The physiology and circuitry associated with dorsal cochlear nucleus neurons (DCN) have been well described. The ability to remotely manipulate neuronal activity in these neurons would represent a step forward in the ability to understand the specific function of DCN neurons in hearing. Although, optogenetics has been used to study the function of pathways in other systems for several years, in the auditory system only neurons in the auditory cortex have been studied using this technique. Adeno-associated viral vectors with either channelrhodopsin-2 fused with GFP (ChR2-GFP) or halorhodopsin fused with mCherry (HaloR-mCherry), capable of expressing light sensitive cation channels or chloride pumps, respectively, were delivered into the dorsal cochlear nucleus (DCN). One to 18 months later, expression of ChR2 and HaloR was observed throughout the DCN. Rhodopsin distribution within the DCN was determined to be within several cell types identified based on morphology and location within the DCN. Expression of ChR2-GFP and HaloR-mCherry was found at both the injection site as well as in regions receiving projections from the site. Wavelength appropriate optical stimulation in vivo resulted in neuronal activity that was significantly increased over pre-stimulation levels with no return to baseline levels during the time of the light exposure. We also examined the effects of optically driven neuronal activity on subsequent tone driven responses in the DCN. In the DCN 75% of the 16 electrode sites showed decreased neuronal activity in response to a tone immediately following light stimulation while six percent were decreased following tone stimulation and 19% of the electrode sites showed no change. This is in contrast to tone driven neuronal activity prior to the light exposure in which the majority of electrode sites showed increased neuronal activity. Our results indicate that expression and activation of rhodopsin within neurons involved in auditory processing does not appear to have deleterious effects on hearing even 18 months following expression. In addition, virally targeted rhodopsins may be useful as tract tracers to delineate as well as modulate the activity of pathways and specific neurons. In the future rhodopsins can be targeted to specific subpopulations of auditory neurons. Ultimately, photostimulation may provide a physiologically relevant method for modulating the function of auditory neurons and affecting hearing outcomes. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Vesicular glutamate transporters: spatio-temporal plasticity following hearing loss.

An immunocytochemical comparison of vGluT1 and vGluT3 in the cochlear nucleus (CN) of deafened versus normal hearing rats showed the first example of vGluT3 immunostaining in the dorsal and ventral CN and revealed temporal and spatial changes in vGluT1 localization in the CN after cochlear injury. In normal hearing rats vGluT1 immunostaining was restricted to terminals on CN neurons while vGluT3 immunolabeled the somata of the neurons. This changed in the ventral cochlear nucleus (VCN) 3 days following deafness, where vGluT1 immunostaining was no longer seen in large auditory nerve terminals but was instead found in somata of VCN neurons. In the dorsal cochlear nucleus (DCN), while vGluT1 labeling of terminals decreased, there was no labeling of neuronal somata. Therefore, loss of peripheral excitatory input results in co-localization of vGluT1 and vGluT3 in VCN neuronal somata. Postsynaptic glutamatergic neurons can use retrograde signaling to control their presynaptic inputs and these results suggest vGluTs could play a role in regulating retrograde signaling in the CN under different conditions of excitatory input. Changes in vGluT gene expression in CN neurons were found 3 weeks following deafness using qRT-PCR with significant increases in vGluT1 gene expression in both ventral and dorsal CN while vGluT3 gene expression decreased in VCN but increased in DCN.

Immunolocalization of vesicular glutamate transporters 1 and 2 in the rat inferior colliculus.

The inferior colliculus is a major relay nucleus in the ascending auditory pathways that receives multiple glutamatergic inputs. Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) most often have complementary non-overlapping distributions and can be used to differentiate glutamatergic inputs. The present study therefore examined co-immunolabeling of VGLUT1 and VGLUT2 in three divisions of the rat inferior colliculus. Additional co-immunolabeling of microtubule-associated protein 2 and neuronal class III beta-tubulin provided visualization of neuronal soma and processes and allowed identification of axo-somatic versus axo-dendritic contacts. Results showed numerous VGLUT1 and 2 immunolabeled terminals in the central nucleus, lateral cortex and dorsal cortex. In all three divisions there was little to no co-containment of the two vesicular glutamate transporters indicating a complementary distribution. VGLUT1 made predominantly axo-dendritic connections in the neuropil, while VGLUT2 had many axo-somatic contacts in addition to axo-dendritic contacts. VGLUT2 immunolabeled terminals were numerous on the soma and proximal dendrites of many medium-to-large and large neurons in the central nucleus and medium to large neurons in the dorsal cortex. There were more VGLUT2 terminals than VGLUT1 in all divisions and more VGLUT2 terminals in dorsal and lateral cortices than in the central nucleus. This study shows that VGLUT1 and VGLUT2 differentiate complementary patterns of glutamatergic inputs into the central nucleus, lateral and dorsal cortex of the inferior colliculus with VGLUT1 endings predominantly on the dendrites and VGLUT2 on both dendrites and somas.

Deafness associated changes in two-pore domain potassium channels in the rat inferior colliculus.

Two-pore potassium channels can influence neuronal excitability by regulating background leakage of potassium ions and resting membrane potential. The present study used quantitative real time PCR and in situ hybridization to determine if the decreased activity from deafness would induce changes in two-pore potassium channel subunit expression in the rat inferior colliculus (IC). Ten subunits were assessed with quantitative real-time PCR at 3 days, 3 weeks and 3 months following bilateral cochlear ablation. TASK-1, TASK-5 and THIK-2 showed significant decreases in expression at all three times assessed. TASK-5, relatively specific to auditory neurons, had the greatest decrease. TWIK-1 was significantly decreased at 3 weeks and 3 months following deafness and TREK-2 was only significantly decreased at 3 days. TASK-3, TWIK-2, THIK-1, TRAAK and TREK-1 did not show any significant changes in gene expression. In situ hybridization was used to examine TASK-1, TASK-5, TWIK-1 and THIK-2 in the central nucleus, dorsal cortex and lateral (external) cortex of the IC in normal hearing animals and at 3 weeks following deafening. All four subunits showed expression in neurons throughout IC subdivisions in normal hearing rats, with TASK-5 having the greatest overall number of labeled neurons. There was no co-localization of subunit expression with glial fibrillary acidic protein immunostaining, indicating no expression in glia. Three weeks following deafening there was a significant decrease in the number of neurons expressing TASK-1 and THIK-2 in the IC, while TASK-5 had significant decreases in the central nucleus and dorsal cortex and TWIK-1 in the lateral and dorsal cortices.

Benzodiazepine receptor ligands are elevated in an animal model of hepatic encephalopathy: relationship between brain concentration and severity of encephalopathy.

Elevated levels of benzodiazepine receptor agonists are found in both animal models of hepatic encephalopathy and in humans with this syndrome. The present study investigated the relationship between agonist levels and the severity of the encephalopathy, as well as the potential reversibility of the syndrome by benzodiazepine receptor antagonists. The concentrations of benzodiazepine receptor ligands in rat brains were measured at several intervals during the induction of liver failure with thioacetamide. Six hours after the first dose of thioacetamide, brain concentrations of benzodiazepine receptor ligands were increased and open field activity decreased compared to control rats. However, the brain concentrations of benzodiazepine receptor ligands correlated better with the stage of hepatic encephalopathy than time after initiation of thioacetamide treatment. The benzodiazepine receptor ligands Ro 15-3505, Ro 15-4513 and CGS-8216 ameliorated motor abnormalities in rats with stage 3 hepatic encephalopathy. Only Ro 15-3505 improved motor activity in rats in stage 2 encephalopathy to levels observed in rats with stage 1 encephalopathy. Furthermore, although Ro 15-4513 and CGS 8216 significantly increased motor activity in stage 4 hepatic encephalopathy, this may reflect their partial inverse agonist properties. These findings support the hypothesis that increased brain levels of benzodiazepine receptor agonists contribute to the severity of hepatic encephalopathy and suggest that high-affinity benzodiazepine receptor antagonists are efficacious in reversing this syndrome.