Immunoreagents.

<p>LM: light microscopy; EM: electron microscopy.</p

SyPhy immunoreactivity in AVCN detected under the electron microscope.

<p>(A) Staining with DAB already indicated that presynaptic profiles were not quantitatively recognized. Black arrows point to SCZs of immunopositive (SyPhy+) synapses, white arrows point to immunonegative (SyPhy-) synapses, always pointing from the postsynaptic side to an AZ; d  =  dendritic profile. (B) Immunogold staining (black particles) confirmed finding illustrated in A; same conventions and scale length. (C) Combining SyPhy immunolabeling with anterograde axonal transport of BDA from cochlear to AVCN proved that primary sensory synapses of various size do not dependably contain SyPhy, with nearby presynaptic profiles massively labeled for SyPhy through immunogold staining. Black arrows point to SCZs of a BDA-positive (co.aff.) or a SyPhy-positive synapses, white arrows point to an SCZ of a synapse negative for both BDA and SyPhy; s  =  neuronal soma. (D) Immunogold (SyPhy, black particles) and DAB (ChAT, white asterisks) double staining, indicating absence of interference between both procedures. Scale bar  = 1 µm for (A) – (D).</p

EPTA stains all types of SCZs.

<p>(A) EPTA staining does not interfere with immunogold detection, as shown here for SyPhy immunoreactivity, also proving that SyPhy positive synapses (SyPhy+) are readily recognized in EPTA treated material; arrows point to SCZs from the postsynaptic side. Scale bar  = 1 µm. (B) Unlike SyPhy immunoreactivity, EPTA does not fail to detect primary sensory endings (co.aff.) indicated by gold-detection of BDA transported into AVCN after intracochlear injection; p  =  presynapse, d  =  dendrite. Arrows point to SCZs from the postsynaptic side, arrowhead indicates presynaptic protrusions of an SCZ. Scale bar  = 0.5 µm. (C) Glutamate immunogold staining (Glu+) demonstrated in conjunction with EPTA at POD7, indicating that not all glutamatergic synapses have disappeared from AVCN after cochlear nerve degeneration. Arrows point to SCZ from the dendritic side, arrowheads indicate presynaptic protrusions. Scale bar  = 1 µm. (D) ChAT immunogold labeling (ChAT+) combined with EPTA staining at POD7; note that the ChAT-positive nerve ending carries an immature SCZ (asterisk), whereas a nearby ChAT-negative synaptic contact (white arrow) shows a fully differentiated EPTA staining including presynaptic protrusions (arrowheads). Scale bar  = 1 µm. (E) Gephyrin immunogold labeling (black particles, Geph+) combined with EPTA staining (dark gray). Arrow points to SCZ from the dendritic (d) side, arrowheads indicate presynaptic (p) protrusions. Scale bar  = 0.5 µm. (F) GAP-43 immunogold labeling (GAP-43+) combined with EPTA staining. Again, arrow points to SCZ from the dendritic (d) side, arrowhead indicates presynaptic (p) protrusions. Scale bar  = 0.5 µm.</p

EPTA stained SCZs in AVCN of the normal adult rat.

<p>(A) Stained profiles appear dark against an otherwise unstained or grainy background and show, when cut perpendicularly, a characteristic morphology, including dense staining of the postsynaptic specialization, some of which marked by an arrow from the postsynaptic side, and, on the presynaptic side (p), ‘peaks’, ‘projections’, or ‘protrusions’ (arrowheads). When tangentially cut (lower left), these projections appear as a grid of patches. d  =  dendritic profile, scale bar  = 1 µm. (B) Section stained with OsO₄ only, showing ultrastructural features such as synaptic vesicles (asterisks) and SCZs (arrows). (C) Section treated as in (B) but with an additional incubation with EPTA revealing presynaptic protrusions (arrowheads), representative of similar frames all failing to show SCZs stained by osmium only. Scale bar for B and C = 0.5 µm.</p

Quantification and ultrastructure of GAP-43 positive profiles in AVCN.

<p>(A) Semi-thin section stained for GAP-43 immunoreactivity. (A') Frame shown in A rendered for computer-based image analysis and quantification. Scale bar  = 20 µm. (B) Ipsilateral-to-contralateral ratio of GAP-43 positive profiles in controls and after 7 and 70 days of cochlear ablation. The right-most column shows counts after correction (corr) for tissue shrinkage to indicate the relative total of GAP-43 positive profiles present by POD70. (C) Ultrastructure of a synaptic contact by POD7 showing rich immunogold staining for GAP-43 typically located inside the presynaptic ending, avoiding occupation of SCZs which is here distinctly immature as revealed by EPTA staining (arrow). Scale bar  = 0.5 µm. (D) Another synapse present by POD7 with a more mature contact zone (arrow, arrowhead points to row of presynaptic protrusions) and correspondingly less GAP-43 immunogold labeling. Scale bar  = 0.5 µm. (E) Fraction of GAP-43 positive synaptic contacts among all SCZs present by POD7 on the lesioned side (i). Numeric triplets below columns as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023686#pone-0023686-g001" target="_blank">Fig. 1</a>.</p

Anterograde tracing and SyPhy immunoreactivity in AVCN.

<p>(A) Anterograde axonal tracing with BDA from cochlea to AVCN, resulting in a great number of fiber segments and presynaptic endings darkly labeled (inset, arrows). Scale bar  = 200 µm, 20 µm for inset. (B) Pattern of SyPhy immunoreactivity and (B') rendering of staining for computer-based image analysis and counting of stained subcellular structures qualified by size and distribution to be presynaptic boutons (arrows). Scale bar  = 20 µm. (C) As must be expected, the number of such boutons is balanced between left and right side (control rat l/r) in normal hearing animals (left). Surprisingly, the balance persisted between ablated and unaffected side (right) by POD7, suggesting that SyPhy immunoreactivity does not reflect deafferentation-dependent changes taking place in the cochlear nucleus. Numeric triplets below columns indicate numbers of fields analyzed, tissue probes (embedded sections), and brains, respectively, on which statistical analysis is based.</p

Quantification of EPTA profiles (all SCZs) and gephyrin positive profiles (inhibitory SCZs) before and at various times after cochlear ablation.

<p>(A) Progression of SCZ density after sensory deafferentation in the ipsilateral (i) AVCN. (B) Balance of ipsilateral-to-contralateral (i/c) ratio of EPTA stained SCZs; in controls left-to-right (l/r) ratios are given. When corrected for lesion-dependent tissue shrinkage (corr), the total number of SCZs present 10 weeks after cochlear ablation can be estimated (right column). (C) Density of gephyrin-positive profiles before and after cochlear ablation. (D) Fractions of gephyrin labeled SCZs among the population of all EPTA labeled SCZs present at pre- and postoperative stages. Numeric triplets below columns as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023686#pone-0023686-g001" target="_blank">Fig. 1</a>.</p

Composition of the population of SCZs in adult controls, by 7 and by 70 days following cochlear ablation.

<p>A deafferentation induced reorganization of the neuronal network in AVCN includes a transient rise of inhibitory synapses and the emergence of GAP-43 positive synaptic contacts. After 10 weeks excitatory synaptic contacts have increased in relative number, apparently due to an extended fraction of cholinergic synapses. ‘∼’ translates to ‘suspected to be’.</p

Video_3.mp4

<p>Neuron–glia interactions contribute to tissue homeostasis and functional plasticity in the mammalian brain, but it remains unclear how this is achieved. The potential of central auditory brain tissue for stimulation-dependent cellular remodeling was studied in hearing-experienced and neonatally deafened rats. At adulthood, both groups received an intracochlear electrode into the left cochlea and were continuously stimulated for 1 or 7 days after waking up from anesthesia. Normal hearing and deafness were assessed by auditory brainstem responses (ABRs). The effectiveness of stimulation was verified by electrically evoked ABRs as well as immunocytochemistry and in situ hybridization for the immediate early gene product Fos on sections through the auditory midbrain containing the inferior colliculus (IC). Whereas hearing-experienced animals showed a tonotopically restricted Fos response in the IC contralateral to electrical intracochlear stimulation, Fos-positive neurons were found almost throughout the contralateral IC in deaf animals. In deaf rats, the Fos response was accompanied by a massive increase of GFAP indicating astrocytic hypertrophy, and a local activation of microglial cells identified by IBA1. These glia responses led to a noticeable increase of neuron–glia approximations. Moreover, staining for the GABA synthetizing enzymes GAD65 and GAD67 rose significantly in neuronal cell bodies and presynaptic boutons in the contralateral IC of deaf rats. Activation of neurons and glial cells and tissue re-composition were in no case accompanied by cell death as would have been apparent by a Tunel reaction. These findings suggest that growth and activity of glial cells is crucial for the local adjustment of neuronal inhibition to neuronal excitation.</p

Image_2.tif

<p>Neuron–glia interactions contribute to tissue homeostasis and functional plasticity in the mammalian brain, but it remains unclear how this is achieved. The potential of central auditory brain tissue for stimulation-dependent cellular remodeling was studied in hearing-experienced and neonatally deafened rats. At adulthood, both groups received an intracochlear electrode into the left cochlea and were continuously stimulated for 1 or 7 days after waking up from anesthesia. Normal hearing and deafness were assessed by auditory brainstem responses (ABRs). The effectiveness of stimulation was verified by electrically evoked ABRs as well as immunocytochemistry and in situ hybridization for the immediate early gene product Fos on sections through the auditory midbrain containing the inferior colliculus (IC). Whereas hearing-experienced animals showed a tonotopically restricted Fos response in the IC contralateral to electrical intracochlear stimulation, Fos-positive neurons were found almost throughout the contralateral IC in deaf animals. In deaf rats, the Fos response was accompanied by a massive increase of GFAP indicating astrocytic hypertrophy, and a local activation of microglial cells identified by IBA1. These glia responses led to a noticeable increase of neuron–glia approximations. Moreover, staining for the GABA synthetizing enzymes GAD65 and GAD67 rose significantly in neuronal cell bodies and presynaptic boutons in the contralateral IC of deaf rats. Activation of neurons and glial cells and tissue re-composition were in no case accompanied by cell death as would have been apparent by a Tunel reaction. These findings suggest that growth and activity of glial cells is crucial for the local adjustment of neuronal inhibition to neuronal excitation.</p