Noise Exposure Alters Glutamatergic and GABAergic Synaptic Connectivity in the Hippocampus and Its Relevance to Tinnitus.

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.

Automated Macro Approach to Quantify Synapse Density in 2D Confocal Images from Fixed Immunolabeled Neural Tissue Sections.

This chapter describes an ImageJ/Fiji automated macro approach to estimate synapse densities in 2D fluorescence confocal microscopy images. The main step-by-step imaging workflow is explained, including example macro language scripts that perform all steps automatically for multiple images. Such tool provides a straightforward method for exploratory synapse screenings where hundreds to thousands of images need to be analyzed in order to render significant statistical information. The method can be adapted to any particular set of images where fixed brain slices have been immunolabeled against validated presynaptic and postsynaptic markers.

GABA is localized in dopaminergic synaptic vesicles in the rodent striatum.

Recently, electrophysiological evidence was given for inhibitory postsynaptic responses at dopaminergic striatal synapses. These responses were independent of the vesicular GABA transporter, VGAT, but dependent on the vesicular dopamine transporter VMAT2. The identity and the exact source of the released molecule, as well as the presence of the putative inhibitory transmitter in VMAT2 containing synaptic vesicles remain to be shown. To clarify this, in particular to determine whether GABA is responsible for the inhibitory response at dopaminergic synapses, we used the electron microscopic immunogold method to label in vivo perfusion fixed striatal tissue with antibodies recognising GABA, VGAT, VMAT2 and tyrosine hydroxylase. We show that about 13 % of tyrosine hydroxylase positive and 11 % of VMAT2 axonal terminals in the caudo-putamen contain significant labelling for GABA. Immunogold signals for tyrosine hydroxylase and VGAT was totally segregated into different pools of nerve terminals. Quantitative analyses of the distance between gold particles signalling GABA and synaptic vesicles showed that GABA was as closely associated with synaptic vesicles in tyrosine hydroxylase positive as in tyrosine hydroxylase negative nerve terminals. Likewise, in dopaminergic terminals GABA and VMAT2 immunogold particles showed a close spatial localization, strongly suggesting the presence of GABA in VMAT2 positive synaptic vesicles. Our results suggest that GABA is exocytosed together with dopamine from dopaminergic nerve terminals in the caudo-putamen through VGAT negative and VMAT2 positive synaptic vesicles.

Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks.

The aim of this study was to clarify the saturation processes of excitatory and inhibitory synapse densities during the long-term development of cultured neuronal networks. For this purpose, we performed a long-term culture of rat cortical cells for 35 days in vitro (DIV). During this culture period, we labeled glutamatergic and GABAergic synapses separately using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of γ-aminobutyric acid (VGAT). The densities and distributions of both types of synaptic terminals were measured simultaneously. Observations and subsequent measurements of immunofluorescence demonstrated that the densities of both types of antibody-labeled terminals increased gradually from 7 to 21-28 DIV. The densities did not show a further increase at 35 DIV and tended to become saturated. Triple staining with VGluT1, VGAT, and microtubule-associated protein 2 (MAP2) enabled analysis of the distribution of both types of synapses, and revealed that the densities of the two types of synaptic terminals on somata were not significantly different, but that glutamatergic synapses predominated on the dendrites during long-term culture. However, some neurons did not fall within this distribution, suggesting differences in synapse distribution on target neurons. The electrical activity also showed an initial increase and subsequent saturation of the firing rate and synchronized burst rate during long-term culture, and the number of days of culture to saturation from the initial increase followed the same pattern under this culture condition.

Synaptotagmin-2 is a reliable marker for parvalbumin positive inhibitory boutons in the mouse visual cortex.

BACKGROUND: Inhibitory innervation by parvalbumin (PV) expressing interneurons has been implicated in the onset of the sensitive period of visual plasticity. Immunohistochemical analysis of the development and plasticity of these inhibitory inputs is difficult because PV expression is low in young animals and strongly influenced by neuronal activity. Moreover, the synaptic boutons that PV neurons form onto each other cannot be distinguished from the innervated cell bodies by immunostaining for this protein because it is present throughout the cells. These problems call for the availability of a synaptic, activity-independent marker for PV+ inhibitory boutons that is expressed before sensitive period onset. We investigated whether synaptotagmin-2 (Syt2) fulfills these properties in the visual cortex. Syt2 is a synaptic vesicle protein involved in fast Ca(2+) dependent neurotransmitter release. Its mRNA expression follows a pattern similar to that of PV throughout the brain and is present in 30-40% of hippocampal PV expressing basket cells. Up to now, no quantitative analyses of Syt2 expression in the visual cortex have been carried out. METHODOLOGY/PRINCIPAL FINDINGS: We used immunohistochemistry to analyze colocalization of Syt2 with multiple interneuron markers including vesicular GABA transporter VGAT, calbindin, calretinin, somatostatin and PV in the primary visual cortex of mice during development and after dark-rearing. CONCLUSIONS/SIGNIFICANCE: We show that in the adult visual cortex Syt2 is only found in inhibitory, VGAT positive boutons. Practically all Syt2 positive boutons also contain PV and vice versa. During development, Syt2 expression can be detected in synaptic boutons prior to PV and in contrast to PV expression, Syt2 is not down-regulated by dark-rearing. These properties of Syt2 make it an excellent marker for analyzing the development and plasticity of perisomatic inhibitory innervations onto both excitatory and inhibitory neurons in the visual cortex.

Neuronal cotransport of glycine receptor and the scaffold protein gephyrin.

The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of gamma-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR-gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein-gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde gephyrin recruitment. Our data suggest a GlyR-gephyrin-dynein transport complex and support the concept that gephyrin-motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of synaptic strength.