Redistribution of synaptic AMPA receptors at glutamatergic synapses in the dorsal cochlear nucleus as an early response to cochlear ablation in rats

Hearing Research

Hearing Research 216–217 (2006) 154–167

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Research paper

Redistribution of synaptic AMPA receptors at glutamatergic synapses in the dorsal cochlear nucleus as an early response to cochlear ablation in rats Maria E. Rubio

*

Department of Physiology and Neurobiology, University of Connecticut, 75 North Eagleville Road, Storrs, CT 06269-3156, United States Received 30 October 2005; received in revised form 2 March 2006; accepted 8 March 2006 Available online 27 April 2006

Abstract This study investigated whether unilateral deafferentation of the presynaptic neuron is key in the control of morphology and the subunit composition and expression of AMPA type glutamate receptors (GluRs) in neurons of the dorsal cochlear nucleus (DCN). Data showed that there are morphological changes at the postsynaptic sites which precede presynaptic changes at the auditory nerve (AN) synaptic ending in response to peripheral damage, in particular that the postsynaptic densities (PSD) of the AN on fusiform cells (FC) are thicker after denervation. Moreover, GluR2, GluR3 and GluR4 AMPA receptor subunits were redistributed, not only at the synapse of FCs receiving direct contact with the AN, but also at the glutamatergic synapse of the parallel fibers on FC and on cartwheel cells (CwC) which are indirectly innervated by the AN. Interestingly, the same synapses in the DCN contralateral to the lesion and with a normal AN synaptic input also redistributed AMPA receptor subunits at the synapse in respond to deafferentation. In these synapses, there was an increase of immunogold labeling for GluR2/3 subunits but not for GluR2 at 2 days after deafferentation.  2006 Elsevier B.V. All rights reserved. Keywords: Cochlear ablation; Rat; Electron microscopy; Postembedding immunogold labeling; AMPA receptors

1. Introduction Electrical activity modifies neuronal signaling at synapses and plays multiple roles in the expression of synaptic plasticity (Bliss and Collingridge, 1993). Synaptic activity may regulate the efficacy of transmission and can also modulate protein content at the synapse, altering the complement of neurotransmitter receptors and downstream signaling machinery (Rao and Craig, 1997; Shen and Meyer, 1999). The postsynaptic response may be deterAbbreviations: ABR, Auditory brainstem response; AMPA, a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA receptors; AN, auditory nerve; CwC, Cartwheel cells; CN, cochelar nuclei; DCN, dorsal cochlear nucleus; FC, Fusiform cells; GluRs, glutamate receptors; PF, parallel fibers; PSD, postsynaptic density; TEM, transmission electron microscopy * Tel.: +1 860 486 9032; fax: +1 860 486 3303. E-mail address: [email protected]. 0378-5955/$ - see front matter  2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.03.007

mined by the number and density of postsynaptic receptors, which in turn may be related to the size of the postsynaptic density (PSD; Nusser, 2000). Moreover, the PSD is itself a plastic structure that is significantly influenced by the amount of activity in the presynaptic element. The response of auditory neurons to acoustic stimulation must maintain rapid transmission and maximize temporal fidelity through their synaptic networks. Consistent with these demands is the observation that targets of the auditory nerve (AN) often express fast kinetic receptors in the cochlear nuclei (CN). Glutamate mediates fast excitatory neurotransmission in the auditory pathway through the activation of AMPA glutamate receptors (Trussell, 1999; Davis and Young, 2000). AMPA receptors (AMPARs) are tetrameric complexes composed of homologous or heterologous subunits (Hollman and Heinemann, 1994) widely expressed in the CN (Trussell, 1999; Petralia et al., 2000). Studies have shown that the type, amount and organization of glutamate

M.E. Rubio / Hearing Research 216–217 (2006) 154–167

receptors (GluRs) at the AN excitatory postsynaptic specializations are critical for efficient transmission of the auditory information in the CN (Rubio and Wenthold, 1997, 1999; Wang et al., 1998; Gardner et al., 1999, 2001). In the DCN fusiform cells (FC) are innervated by both the glutamatergic, myelinated AN fibers and unmyelinated parallel fibers (PF; Fig. 1A). Remarkably, the AN fibers target the AMPA GluR4 subunit on the basal postsynaptic dendrites (Rubio and Wenthold, 1997, 1999). Other AMPA receptor subunits like the GluR2, and GluR3 are present at both synapses (Rubio and Wenthold, 1997, 1999). The presence of the GluR2 subunit indicates that both synapses are Ca2+ impermeable (Gardner et al., 2001). This result suggests that innervation plays an instructive role on gene expression, and that mechanisms for subcellular targeting of different GluR subunits are coordinated in an undetermined manner by particular presynaptic neurons (Lawrence and Trussell, 2000). It is not well understood, however, whether the AN itself and/or the levels of AN synaptic activity are involved in the appropriate expression and/or distribution of GluRs at the glutamatergic synapse and on CN neurons. Changes in the efficiency of synaptic transmission appear to be a key factor in the modifications of connections between neurons. One way in which this efficiency can be modified is through alterations of the properties and number of neurotransmitter receptors at the synapse (Bear and Malenka, 1994; Malinow and Malenka, 2002). Therefore, any modification in the composition of GluRs complexes at the synapse, after cochlear

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trauma and/or under abnormal auditory signaling, will likely change the CN neuronal responses. Any change in their neuronal responses will have profound effects on the responses of downstream cells. Deafferentation has been used as a tool to analyze aberrant neuronal behavior and plasticity in the auditory pathway (Potashner et al., 2000; Chang et al., 2002). Therefore, if cochlear ablation abolish the activity of the AN, it is likely that CN neurons will respond to the lack of acoustic stimulation by changing the expression of the excitatory receptors. Biochemical binding studies have shown that the absence of auditory input affects the amount and activity of GluRs in CN (Suneja et al., 2000). It has not been determined, however, whether these changes are in response to a redistribution of GluR subunits at the AN synapse, and whether deafferentation affects the expression and subunit composition of GluRs at the AN itself and/or other excitatory synapses on CN neurons. To directly address this lack of information, this study combined unilateral cochlear ablation procedures with quantitative postembedding immunogold labeling at the electron microscopy level. This study focused on the analysis of the expression and redistribution of the GluR2–4 AMPAR subunits that are relevant for the kinetics of the channel (Trussell, 1999) and are expressed in FC glutamatergic synapses, on the excitatory synaptic circuitry of the FC in the DCN. Results show that deafferentation remodels the postsynaptic membranes in apposition to the AN of the lesion side as an early response to deprivation of the AN synaptic input. Further, there is also a reapportionment of AMPAR subunits at glutamateric synapses in the DCN of the ipsilateral and contralateral side to the lesion. 2. Material and methods 2.1. Animals A total of nine postnatal day 22 Sprague–Dawley rats were used in these experiments. The animals were separated in one control group (n = 2) and one experimental group (two animals for 4 h, three for 2 days and two for 1 week survival after unilateral cochlear ablation). The handling and care of the animals prior to and during the experimental procedures were approved and supervised by the University of Connecticut IACUC and followed NIH guidelines. 2.2. Anesthesia

Fig. 1. (A) Schematic of the excitatory synaptic circuitry on FC. AN: auditory nerve on basal dendrites; PF: parallel fibers of the granule cells (GC) synapsing on apical dendrites. The intrinsic inhibitory input of the cartwheel cells (CwC) on apical dendrites is also represented. PF synapse on CwC. The circles highlight the synapses analyzed. To simplify the diagram other excitatory inputs on GC and other inhibitory inputs on FC are not represented. (B) ABR recordings in normal hearing animals and after unilateral cochlear ablation. (C and D) Hematoxylin and eosin staining of the rat temporal bone and bulla contralateral (C) and ipsilateral (D) to the lesion (*: ablated cochlea; arrow heads: ganglia).

A mixture of ketamine (60 mg/kg body weight) and xylazine (6.5 mg/kg body weight) were injected intramuscularly to induce deep anesthesia before auditory brainstem response (ABR) testing, unilateral cochlear ablation and transcardial perfusion. 2.3. ABR test Animals were anesthetized as described above and ABRs were recorded in response to a series of 500 clicks

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presented separately to each ear. Three subcutaneous pin electrodes were placed on the rat’s head at the left ear, right ear and vertex (top of head). Electrical activity evoked by repetitive closed-field acoustic stimuli (1 ms clicks) was amplified (AC differential amplifier, 1000·, passband 10 Hz to 3 kHz), digitized (12-bit A/D, 50 kHz sampling rate) and averaged. In closed-field stimulation, the resulting ABR was used prior to cochlear ablation to confirm that both ears were functional. After cochlear ablation at 4 h and 2 and 7 days, the ABR was used to confirm that hearing on the ablated side was eliminated (Fig. 1B). 2.4. Unilateral cochlear ablation After the ABR test, and while still under deep anesthesia, seven animals were put on a warm blanket under a stereomicroscope. An incision was then made in the skin (around the pinna) to expose the external auditory canal and the tympanic membrane. The head of the animal was oriented so as to visualize the tympanic membrane, which was then broken and the malleus and incus subsequently removed with a fine rongeurs. Right cochleas were ablated mechanically by crushing with a forceps and aspirating the remains. The inner ear cavity was cleaned with sterile saline and filled with gel foam. The surgical area was rinsed with saline, the skin incision closed with sutures and disinfected. Animals were kept on a warm blanket until they awoke. At 4 h, 2 and 7 days post surgery animals were transcardially perfused with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.12M phosphate buffer pH 7.2. After, brains were removed, fixed for an additional hour at 4 C, rinsed in buffer and stored overnight at 4 C. Brainstems were sliced on a vibratome. Half of the slices were processed for conventional transmission electron microscopy (TEM) and were postfixed in 1% osmium tetroxide, washed and dehydrated in ethanol, infiltrated with epoxy resins and flat-embedded (Rubio and Juiz, 1998). The other half was processed for freeze-substitution technique followed by postembedding immunogold labeling (see below). To evaluate the degree of cochlear damage, contralateral

and ipsilateral to the lesion temporal bones were decalcified in EDTA, cryoprotected, cryosectioned and stained with haematoxylin and eosin (Fig. 1C and D). 2.5. Freeze substitution and postembedding immunogold labeling DCN vibratome sections were processed for freeze-substitution and low-temperature embedding as previously described (Rubio and Wenthold, 1997; Rubio and Juiz, 2004). Postembedding immunogold labeling of GluR2, GluR3 and GluR4 AMPA receptor subunits followed a protocol similar to that described by Rubio and Wenthold (1997). Polyclonal affinity purified antibodies for GluR2/3 and GluR4 (2 lg/ml; Wenthold et al., 1992) and one monoclonal affinity-purified antibody for GluR2 that recognizes the N-terminus of the GluR2 subunit (1:300; Chemicon, Temecula) were used. Double postembedding immunogold labeling for AMPA receptor subunits and GABA (1:400; Chemicon) was performed as previously described (Rubio and Juiz, 2004). Primary antibodies were labeled with 5 nm (AMPA) or 10 nm (GABA) colloidal gold-coupled secondary antibodies (Amersham; Piscataway). Controls were carried out by omitting the primary antibody in the immunogold labeling. Electron micrographs were taken at 34,300· magnification with a Philips 300M TEM and scanned at a resolution of 1600 dpi using an Epson Expression 1680 scanner. Immunostaining was also analyzed with a TECNAI 12 Biotwin TEM. The images were captured with an AMT CCD camera at 49,000· magnification. Image processing was performed with Adobe Photoshop by using only the brightness and contrast commands to enhance gold particles. Postembedding immunogold labeling of the ipsilateralablation, contralateral sides and normal-hearing littermates was performed simultaneously. Only well-identified synapses were included in the analysis. Table 1 shows the number of synapses analyzed for normal hearing littermates and experimental animals for each of the subunits analyzed.

Table 1 Number of synapses analyzed after postembedding immunogold in normal hearing and deafferentated animals at 4 h and 2 days after cochlear ablation Normal (n = 2) AN/FC

PF/FC

Single immuno-gold labeling GluR2 30 30 GluR2/3 30 30 GluR4 30 30 Double immuno-gold labeling GluR2 15 15 GluR2/3 15 15 GluR4 15 –

4 h (n = 2) PF/CW

AN/FC

2 days (n = 3) PF/FC

AN/FC

PF/FC

PF/CW

Ipsi.

Contra.

Ipsi.

Contra.

Ipsi.

Contra.

Ipsi.

Contra.

Ipsi.

Contra.

30 30 –

35 35 30

44 30 30

40 38 50

35 35 40

35 34 35

30 34 32

35 40 45

35 35 45

50 50 –

50 50 –

15 15 –

15 15 15

15 15 15

15 15 15

15 15 15

15 15 15

15 15 15

20 20 15

15 15 15

20 20 –

15 15 –

Single postembedding immunogold labeling: average number of synapses analyzed per animal = 15–20. Double postembedding immunogold labeling: average number of synapses analyzed per animal = 7–10. Average mean did not differ between or among the animals analyzed per each of the DCN analyzed in each group. n, number of animals analyzed; Ipsi., ipsilateral side to the lesion; Contra, contralateral side to the lesion.

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Fig. 2. Deafferentation affects the AN synaptic button. Electronmicrographs show EPON embedded tissue of the AN synapse on basal dendrites of FC in normal and at 2 and 7 days after cochlear ablation. (A) Electron micrograph shows an AN synapse on basal dendrites of FC of a normal hearing animal. (B) AN myelinated fibers (ANf) in the deep DCN 2 days after ablation. Axoplasm presents large vacuoles that are sign of early stages of degeneration. (C and D) AN synaptic button on a basal dendrite of FC at 2 days after deafferentation. Granulomatose electrondense material is observed among synaptic vesicles (white asterisk). Synaptic endings also show larger dense round and coated synaptic vesicles (arrows). (E) AN synapse on a basal dendrite of FC at 7 days after cochlear ablation. The synaptic vesicles looked larger and compacted. A large endocytotic vesicle is observed in the FC dendrite (*). The PSD lacks of the characteristic electron-dense material. Scale bar: 0.25 lm.

2.6. Identification of FC, CwC and excitatory synapses in the DCN at the TEM level The procedure to identify FC and their apical and basal dendrites was similar to that used in Rubio and Wenthold (1997) and Rubio and Juiz (2004).

2.6.1. AN synapses on FCs (Figs. 2–4) The AN synapses are made on basal dendrites of FC (Smith and Rhode, 1985; Zhang and Oertel, 1994; Rubio and Wenthold, 1997). These synaptic endings were identified using the following criteria (Kane, 1974; Smith and Rhode, 1985; Ryugo and May, 1993; Rubio and Wenthold,

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Fig. 3. Deafferentation affects the thickness of the PSD of the AN synapse. (A) Electron micrograph shows an AN synapse on basal dendrites of FC of a normal hearing animal. A dashed line has been drawn to better show the extension of the PSD. (B) AN synapses after 4 h of denervation, no changes were observed in the cytoplasm of the AN. Electron micrograph shows a thicker PSD when compared to control (A). The electronmicrograph of the ablated side also shows an invaginated plasma membrane (*). A dashed line has been drawn to better show the extension of the PSD. (C1 and C2) AN synapse on the cell body (C1) and basal dendrites (C2) of FC at 2 days after denervation. A thicker PSD is clearly observed. The internal cytoplasmic portion of the PSD is observed less electrondense that the external portion facing the synaptic cleft and the AN. Postsynaptic dendrites apposed to the AN present endocytotic vesicles (* in C2) A dashed line has been drawn to better show the extension of the PSD. Scale bar for A–C: 0.25 lm. (D) Histogram shows the average thickness of the PSD (±SEM) of the AN on basal dendrites of FC in control (n = 2 animals; PSDs n = 30) and 4 h (n = 2 animals; PSDs n = 30) after ablation (*p < 0.01).

1997; Rubio and Juiz, 2004): (1) location in the nucleus (the AN synapses are distributed in the fusiform and deep layers of the DCN); and (2) ultrastructural characteristics. The AN synapses are large presynaptic endings (average size of 2.3 ± 1.1 lm) which have numerous large, clear, and round synaptic vesicles (39.8 ± 6.1 nm, mean ± SD; in diameter), and contain numerous mitochondria. They form multiple asymmetric (Gray I) synaptic contacts and contain attachment plaques or ‘‘puncta adherentia’’.

2.6.2. Parallel fibers synapses on FCs (Fig. 5) Excitatory synaptic inputs from parallel fibers (PF) are the predominant synaptic population on apical dendrites of FC and the only excitatory input to these dendrites (Smith and Rhode, 1985; Oertel and Wu, 1989; Manis, 1989; Osen et al., 1995; Rubio and Wenthold, 1997; Rubio and Juiz, 2004). PF synapses were identified using wellestablished criteria by Kane (1974), Mugnaini (1985), Rubio and Wenthold (1997) and Rubio and Juiz (2004),

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Fig. 4. Deafferentation affects the synaptic expression of GluR2/3, GluR2 and GluR4 at the AN/FC synapse. Electron micrographs show postembedding immunogold labeling (5 nm gold) for GluR2/3 (A1–4), GluR2 (B1–2) and GluR4 (C1–2) at the PSD of the AN on basal dendrites of FC of the ablation side after 4 h and 2 days survival time. Insets show a larger magnification of the PSDs decorated with gold particles. (A1–4) Gold particles for GluR2/3 are observed at the PSD at 4 h (A1) after deafferentation. At 2 days (A2–4) some PSD of the AN present labeling for GluR2/3 but preferentially it redistributes intracellularly (arrows). (B1–2) Gold particles for GluR2 decorating the PSD of the AN are observed at 4 h (B1) and 2 days (B2) after cochlear ablation. (C1–2) Gold particles for GluR4 after 4 h (C1) or 2 days (C2) after cochlear ablation are not observed at the PSD, but are observed intracellularly (arrows). (*): endocytotic vesicles. Scale bar for A–C: 0.25 lm. (D) Histograms show the density of gold particles/lm length of PSD ± SEM for GluR2/3, GluR2 and GluR4 at the PSD of AN on FC of normal hearing animals (control; n = 2) and after 4 h (n = 2) and 2 days (n = 3) after cochlear ablation of the ipsilateral and contralateral side to the lesion (**p < 0.01; ***p < 0.001).

and include (1) Their location in the molecular layer of the DCN (parallel fibers are described as unmyelinated axons that run parallel to the surface of the nucleus); and (2) ultrastructural characteristics. The PF synapses are small synaptic endings, which contain small, clear synaptic vesicles (37.2 ± 6.1 nm mean ± SD; in diameter), and make asymmetrical synaptic contacts (Gray I) onto spines and/ or dendritic shafts of apical dendrites of FCs. 2.6.3. PF synapses on dendritic spines of cartwheel cells (Fig. 6) Cartwheel cells (CwC) were identified following wellestablished criteria by Wouterlood and Mugnaini (1984), Berrebi and Mugnaini (1991) and Rubio and Juiz (2004). Location in the nucleus was also used to identify cell bodies and dendrites. CwC bodies were located between the fusiform and molecular layer and most of the dendritic spines analyzed were located in the most apical 100 lm of the

DCN (this measurement was estimated based on grid hole width). Dendritic spines of CwC are abundant in the molecular layer and are easily recognized because they are large, they have an elliptical shape and are enriched with membranes of the smooth endoplasmic reticulum. 2.7. Identification of FC, CwC and excitatory synapses by double postembedding immunogold labeling for GABA and AMPA receptors I performed a second study to identify FC and CwCs profiles and their excitatory synapses. I used a double postembedding immunogold labeling for GABA and GluR2–4 AMPAR subunits. CwCs are inhibitory neurons that have been shown to be immunopositive for GABA (Osen et al., 1990). Therefore, GABA antibodies can be used to label and identify dendritic profiles of CwCs in the DCN. On the other hand, FC are excitatory neurons

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Fig. 5. Deafferentation affects the synaptic expression of GluR2/3 at the PF/FC synapse. Electron micrographs show postembedding immunogold labeling (5 nm gold) for GluR2/3 (A1–4), GluR2 (B1–3) and GluR4 (C1–3) at PSD of PF on FC in controls, and FC of the ablation side at 4 h and 2 days survival time. (A1–4) Postembedding immunogold labeling for GluR2/3 at PF synapses on apical dendrites of FC in control (A1) and 4 h (A2) and 2 days (A3–4) after lesion. All the PSDs are observed decorated with gold particles. The PSDs after 2 days of ablation (A3–4) show more gold particles than in controls and 4 h. (A4) A putative inhibitory synaptic ending (1) on apical dendrite of FC lacks of gold particles for GluR2/3. Arrows: gold particles labeling the intracellular pool of AMPA receptors. Insets show a larger magnification of the PSDs. (B1–3) Postembedding immunogold labeling for GluR2 shows the PSD of PF on apical dendrites of FC decorated with gold particles. (C1–3) The PSD of the PFs on FCs lacks of gold particles for GluR4. Gold particles are observed intracellularly (arrow). FCd: FC dendrite; FCs: FC spine). Scale bar for A–C: 0.25 lm. (D) Histograms show the density of gold particles/lm length of PSD for GluR2/3 and GluR2 at the PSD of PF on FC of normal hearing animals (control; n = 2) and after 4 h (n = 2) and 2 days (n = 3) of cochlear ablation of the ipsilateral and contralateral side to the lesion (***p < 0.001).

that have been shown immunonegative for GABA (Osen et al., 1990). The glutamatergic synaptic endings of the AN, and PF are immunonegative for GABA (Rubio and Juiz, 2004). In the molecular layer, cartwheel and stellate cells are known to be immunopositive for GABA (Osen et al., 1990). However, one of the main differences between these two cell types at the electron microscopy level, is that the PF endings synapse on the dendritic shaft of stellate cells and not on the dendritic spine, as occurs on CwC (Mugnaini, 1985). Therefore, dendritic spines immunogold labeled for GABA in the molecular layer of the nucleus, were considered from CwC (Fig. 7). FCs are excitatory, and are immunonegative for GABA (Osen et al., 1995; Rubio and Juiz, 2004). In this study, the PF synapses analyzed, lacked of gold particles for GABA (Fig. 7), and were observed synapsing on dendritic spines immunogold

labeled for GABA or on dendritic profiles (spines and shafts) immunonegative for GABA. These synaptic endings shared the same ultrastructural and morphometric parameters than synaptic endings identified as PF after single immunogold labeling for AMPA receptors (Fig. 5; Rubio and Wenthold, 1997, 1999) or glutamate (Rubio and Juiz, 1998, 2004). Synaptic buttons identified as terminals of the AN were observed synapsing on dendritic profiles that lacked gold particles for GABA (Fig. 7) and were located in the fusiform cell layer. These dendrites with the AN synapse were observed emerging from a cell body with similar ultrastructural characteristics as the one previously described for FC (Smith and Rhode, 1985; Rubio and Wenthold, 1997, 1999). The AN synaptic endings lacked also of gold particles for GABA (Fig. 7; Rubio and Juiz, 2004) and shared the same ultrastructural and morphometric parameters than synaptic endings identified as the AN

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Fig. 6. Deafferentation affects the synaptic expression of GluR2/3 and GluR2 at the PF/CwC synapse. Electron micrographs show postembedding immunogold labeling (5 nm gold) for GluR2/3 (A1–2) and GluR2 (B1–2) at the PSD of PF on CwC in controls (A1 and B1) and CwC of the ablation side at 2 days after ablation (A2 and B2). CwCs: cartwheel cell spine; PF: parallel fibers. Arrows indicates intracellular labeling. Insets show a larger magnification of the PSDs. Scale bar: 0.25 lm. (C) Histograms show the density of gold particles/lm length of PSD for GluR2/3 and GluR2 at the PSD of PF on CwC of normal hearing animals (control; n = 2) and after 2 days (n = 3) of deafferentation of the ipsilateral and contralateral side to the lesion (***p < 0.001).

Fig. 7. Double postembedding immunogold labeling for AMPAR subunits (5 nm gold particles) and GABA (10 nm gold particles) in the DCN. Gold particles for GABA are observed decorating synaptic endings containing pleomorphic synaptic vesicles and synapsing on basal (A) and apical (B) dendrites of FC. Neither, of these dendritic profiles (A and B) or the synaptic endings identified as the AN (A) or the PF (B), were observed immunogold labeled for GABA. Insets show a higher magnification of the PSDs. (A) The AN synapse on basal dendrite of FC is observed enriched with gold particles for GluR2/3. (B) The PF synapse on a dendritic spine of FC is observed lacking gold particles for GluR4. (C) A dendritic spine of CwCs on the molecular layer of the DCN is observed immunogold labeled for GABA. A PF synaptic ending lacking of gold particles for GABA, is observed synapsing on a CwC dendritic spine. The PSD is observed enriched with gold particles for GluR2. Scale bar: 0.25 lm.

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after single immunogold labeling for AMPA receptors (Fig. 5; Rubio and Wenthold, 1997, 1999) or glutamate (Rubio and Juiz, 1998, 2004). 2.8. Morphometric analysis of the AN postsynaptic density (PSD) on FCs

among the animals analyzed per each group (control, 4 h and 2 days after unilateral cochlear ablation). The quantitative data presented in this study correspond to the analysis obtained from the single postembedding immunogold labeling for GluR2–4 AMPAR subunits. 3. Results

The average thickness of a PSD was calculated as described by Dosemeci et al. (2001) from well-identified PSDs apposed to AN synapses. Micrographs were enlarged to X82,500 for measurements, which were done blind on coded images in random order with JScion NIH image. To measure the average thickness of a PSD, its cytoplasmic outline of the PSD, including the associated dense material, was traced, and this area was then enclosed by tracing the postsynaptic membrane separately (Dosemeci et al., 2001). The area was then divided by the length of the postsynaptic membrane to derive an average thickness for each PSD for each synapse. Samples included 30 AN synapses on basal dendrites of FC from normally hearing animals and 30 from the ipsilateral-lesion side at 4 h after cochlear ablation. 2.9. Quantitative evaluation of GluR2, GluR2/3 and GluR4 receptor immunolabeling The distribution and relative density of the GluR2, GluR2/3 and GluR4 subunit immunolabeling in the AN and PF was determined for the AN/FC, PF/FC, and PF/ CwC synapses at 4 h and 2 days after cochlear ablation (Table 1). Samples included synapses from the DCN of normally hearing animals and from ipsilateral-ablated and contralateral side to the lesion. The linear density of gold particles at the PSD was computed using JScion NIH image for each PSD profile by dividing the number of gold particles in a PSD profile by the membrane length of that profile. The average linear density was computed across all profiles. Data of the ipsilateral side to the ablation was compared to the contralateral side to the ablation and to normal hearing littermates. Although quantification after double immunogold labeling is not recommended (Rubio and Wenthold, 1999), I calculated the density of gold particles for GluR2/3, GluR2 and GluR4 in all the synapses of interest after the double postembedding immunogold labeling (Table 1, sample included 7–10 synapses per antibody, per each of the synapses of interest, and per animal). The density of gold particles per length of PSD was performed for the single and for the double immunogold labeling, separately. Similar results were obtained with both procedures. 2.10. Statistical analyses The Student’s t-test assuming unequal variance and the ANOVA one single factor test were performed with Microsoft Excel. The average mean of the density of gold particles per antibody and synapse was not found different

3.1. Peripheral injury remodels AN postsynaptic membranes as an early response to denervation Structural and functional reorganization occur in the adult nervous system in response to direct or indirect perturbations, including sensory deprivation (Bilak et al., 1997). This study investigates whether deprivation of the AN synaptic input reorganized synapses receiving direct excitatory innervation of the AN on basal dendrites of FCs in the DCN. Electron microscopic observation of AN synaptic buttons at 4 h after cochlear ablation showed similar ultrastructural characteristics as AN synapses in normal hearing littermates. At 2 days after cochlear ablation, early signs of AN degeneration were observed in myelinated fibers in the deep DCN and at AN synaptic buttons (Fig. 2). The latter presented electrondense granulomatose bodies, an increase in coated dense vesicles, and enlarged synaptic vesicles. Also observed were synaptic vesicles, which appeared to be compacted with less axoplasmic material surrounding them (Fig. 2). The PSD is a plastic structure strongly influenced by the presynaptic element (Gulley et al., 1977; Dosemeci et al., 2001). Therefore, I investigated the early effects of denervation on the PSD and postsynaptic membranes of AN synapses on basal dendrites of FC. Morphological changes at the postsynaptic neuron were already observed at 4 h after cochlear ablation. As shown in Figs. 2 and 3, three distinct responses were seen (1) an increase in the thickness of the PSD; (2) an increase in endocytotic vesicles adjacent to the PSD; and (3) an increase in coated vesicles. A morphometric analysis was performed to determine how much the PSD increased in thickness after the ablation (Fig. 3). The data from a total of 60 PSDs (controls n = 30; unilateral side to the lesion n = 30), showed that there was a 2-fold increase in PSD thickness of the AN/FC synapse on the ipsilateral side to the lesion (Fig. 3). By contrast, the length of the PSDs was similar to that of normal hearing matched-age animals (average 0.2 lm). At 2 days after denervation the increase in thickness was still present, although the cytoplasmic face of the PSD was fluffy and appeared less condensed (Fig. 3C–D). The AN synapses at 1 week after unilateral cochlear ablation were also analyzed (Fig. 2E). Ultrastructural analysis showed that the electrondense material characteristic of the PSD disappeared. These data indicate that there is a fast and early remodeling of the excitatory synapse of the AN on FC in response to denervation. Further, they also show that deafferentation alters earlier the organization of the postsynaptic

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membrane than the structural components of the presynaptic button. 3.2. Redistribution of GluR2/3 and GluR4 subunits at AN synapses on FC after unilateral cochlear ablation In many regions of the CNS, there appears to be a correlation between postsynaptic receptor subtype and the source of synaptic input. In the DCN, the subunit composition of GluRs at the primary synapses in FC is important for a fast and adequate transmission of the acoustic information. In addition, synaptic activity has been shown to influence GluR subunit composition (Liu and Cull-Candy, 2000). Alterations in the auditory processing after AN damage may be caused by physiological changes in excitatory transmission, which might become apparent within minutes of inflicting cochlear damage (Boettcher and Salvi, 1993; Bledsoe et al., 1995; Vale and Sanes, 2002). Nevertheless, it is unknown how central auditory synapses reorganize excitatory synapses in response to deafferentation. It is possible that brainstem auditory neurons change the number or distribution of excitatory receptors to compensate for deficits of AN synaptic activity. In this study, I investigated the early effects of denervation of the AN on the expression and distribution of AMPAR subunits at the AN synapse on basal dendrites of FC. Of particular interest was the capability of the FC to control the subunit composition of GluR2–4 AMPAR subunits in response to deafferentation. Postembedding immunogold labeling after freeze-substitution at the EM level was used to directly assess the changes in receptor expression and distribution at the synapse at 4 h and 2 days after unilateral cochlear ablation. This technique has several advantages: (1) it allows direct visualization of tissue ultrastructure and immunolabeling (gold particles); (2) all available antigen sites on the ultrathin section can be labeled (avoiding problems of poor antibody penetration); (3) the gold particles can be quantified. Using this approach one can determine whether the expression of specific receptor subunits at these locations increases or decreases in response to cochlear damage. Analysis of the postembedding immunogold labeling after 4 h of cochlear ablation showed differences in the distribution and density of gold particles (±SEM gold particles/lm) for GluR2/3 and GluR4 subunits at the PSD of AN synapses on basal dendrites of FC (Fig. 4). Gold labeling for GluR2/3 showed a significant increase (p < 0.01) at the PSD ipsilateral to the lesion (24.5 ± 4.0 vs. normal hearing animals 12.3 ± 3.3). On the other hand, gold particles labeling GluR4 were rarely observed at the PSD and they appeared to be redistributed more intracellularly (Fig. 4). The data after 2 days of cochlear ablation (Fig. 4) showed that gold particles labeling GluR4 were rarely observed at the PSD of the AN. The analysis for GluR2/ 3 subunits revealed that the density of gold particles at the PSD was lower (10.3 ± 2.9) than normal hearing litter-

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mates (12.3 ± 3.3). Although this decrease was not found to be significant, there was a larger number of PSD without gold particles. The analysis showed that the range of gold particles for GluR2/3 in the unlesioned animals was from 2 to 7 with a mean of gold particles/PSD of 5. In the ipsilateral side to the lesion the range obtained was from 0 to 8 particles with a mean of gold particles/PSD of 3.3. These data indicate that there is a selective decrease in the expression of GluR2/3 in some PSD apposed to the AN on basal dendrites of FC after deafferentation. Next, I investigated whether or not the early increase in the expression of the GuR2/3 after 4 h, and the later decrease of these subunits on the 2nd day after deafferentation, was associated with changes in the expression of the GluR2 subunit (Fig. 4). The presence of the GluR2 subunit in the AMPA receptor complexes indicates that the synapses are Ca2+ impermeable (Gardner et al., 2001). Data showed that there was an increase of GluR2 at the PSD of the AN synapse on FC at 4 h after cochlear ablation (21.4 ± 4.9 vs. normal hearing animals 9.4 ± 2.7). This increase was found to be slightly significant (p < 0.05). At 2 days the density of gold particles for GluR2 at the PSD of the AN was found similar to normal hearing littermates (ablated: 10.3 ± 2.9 vs. control: 9.4 ± 2.7). 3.3. Redistribution of GluR2/3 subunits at the PF synapses on FC after unilateral cochlear ablation Granule cells are the main excitatory interneuron in the DCN. They receive the unmyelinated type II fibers of the AN (Brown and Ledwith, 1990) as well as excitatory input from various central auditory and non-auditory nuclei (Weedman et al., 1996 for review; Shore et al., 2000). The axons of the granule cells, the PF, innervate the apical dendrites of FC. Therefore, by analyzing the PF/FC synapse we can determine whether the loss of the AN input after cochlear ablation redistributes AMPA receptor subunits at this synapse. Analysis of the postembedding immunogold labeling for GluR2/3 and GluR4 at the PF/FC synapse at 4 h after cochlear ablation was the same as in normal hearing littermates (Fig. 5) and previous published data (Rubio and Wenthold, 1997). At 2 days after ablation, PF synapses continued lacking gold particles for GluR4. On the other hand, the expression for GluR2/3 was significantly increased (p < 0.001; ablated: 52.4 ± 5.1 SEM gold particles/lm of PSD vs. control 12.7 ± 4.3; Fig. 5). Analysis of the postembedding immunogold labeling showed an increase of GluR2 (21.3 ± 3.9) at 2 days after cochlear ablation; however, this was not found significant when compared to normal hearing littermates (12.7 ± 4.4; Fig. 5). Thus, the increase in GluR2/3 was related to an upregulation of the GluR3 subunit. The PF synapses also innervate the cartwheel cells (CwC) in the molecular layer of the DCN (Wouterlood and Mugnaini, 1984). Through this synapse CwCs receive indirect AN synaptic stimulation to inhibit FCs (Golding

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and Oertel, 1997; Davis and Young, 2000). The PSD of the PF on CwC dendritic spines is enriched with the GluR2 and 3 subunits and has low levels of GluR4 (Petralia et al., 1996; Gardner et al., 2001). Therefore, the PF/ CwC synapse permits one to test whether or not neurons that receive indirect input of a main source of excitation also redistribute AMPAR subunits at the synapse in response to the denervation. Analysis of the postembedding immunogold labeling for GluR2/3 subunits (Fig. 6) revealed a significant increase (p < 0.001) in the gold labeling for these subunits at the PF/CwC synapses at 2 days after cochlear ablation (27.7 ± 3.3 SEM gold particles/lm vs. control: 14.8 ± 3.9). The expression for GluR2 was also found significantly increased (p < 0.001; 14.7 ± 1.8) when compared to normal hearing animals (8.1 ± 0.4; Fig. 6). 3.4. Changes in AMPA receptor expression on the contralateral side The CN is the first site in the central auditory system where convergence of binaural information occurs. Interaction between cochlear nuclei can take place by way of the commissural pathway (Cant and Benson, 2003) or via descending inputs from the superior olivary complex and inferior colliculus (Spangler et al., 1987; Shore et al., 1991). Contralateral excitatory inputs to the CN exist, and evidence showed that they could compensate rapidly for large changes in afferent input (Sumner et al., 2005). To further explore this hypothesis, I examined whether or not the glutamatergic synapses of the AN on FC, and PF on FC and CwC cells of the contralateral side responded to denervation by modifying the expression of the GluR2–4 AMPAR subunits at the synapse. At 4 h after cochlear ablation, postembedding immunolabeling showed that the expression for the GluR2/3 and GluR4 subunits at the AN on FCs was similar to normal hearing littermates (Fig. 4). At 2 days after deafferentation the expression of GluR4 was found unaltered; however, the expression for GluR2/3 was significantly increased (p < 0.001; 40.3 ± 6.8 SEM gold particles/lm of PSD vs. control: 12.7 ± 4.3). The expression for GluR2 (17.4 ± 3.4) was not found significantly increased (p > 0.05) when compared to normal hearing animals (9.4 ± 2.7). The expression of the GluR2/3 AMPA receptor subunits was also analyzed at the PF/FC and PF/CwC synapses of the contralateral side at 4 h and 2 days after cochlear ablation (Figs. 5 and 6). At 4 h, the expression for GluR2/3 at the PSD of both synapses was found similar to normal hearing littermates. But after 2 days of the lesion a significant increase (p < 0.001) in the expression of the GluR2/3 subunits was found at the PF/FC (35.2 ± 4.6 vs. control: 12.7 ± 4.3) and PF/CwC synapses (35.1 ± 4.1 vs. control: 14.8 ± 3.9). The analysis of the postembedding immunogold labeling for the GluR2 subunit at the PF/ FC (15.4 ± 1.9) and PF/CwC (8.5 ± 0.5) synapses was sim-

ilar to normal hearing littermates (PF/FC: 12.7 ± 4.3; PF/ CwC: 8.1 ± 0.4). 4. Discussion This study showed that there are morphological changes at the postsynaptic sites which precede presynaptic changes at the AN synaptic ending in response to peripheral damage, in particular that the PSDs of the AN on FC are thicker after denervation. Moreover, AMPA receptor subunits were redistributed, not only at the synapse receiving direct contact with the AN, but also at other glutamatergic synapses indirectly influenced by the AN. Interestingly, the same synapses in the DCN contralateral to the lesion and with a normal AN synaptic input also redistributed AMPA receptor subunits at the synapse in respond to deafferentation. Thus, molecular rearrangements are a widespread response to loss of sensory input in the DCN. 4.1. Remodeling of the AN postsynaptic membranes as an early response to deafferentation The presynaptic element can influence the size and molecular components of the PSD (Gulley et al., 1977; Dosemeci et al., 2001). In response to denervation, there was an early remodeling of the postsynaptic membranes on basal dendrites of FC beneath the AN ending of the ipsilateral side. During the first two days of denervation, the PSD increased in thickness, its cytoplasmic face appeared less condensed and the adjacent cytoplasmic membrane presented an increase in the number of endocytotic vesicles. Similar early changes were described in the AVCN after unilateral cochlea ablation (Gulley et al., 1977). In the CNS, previous studies had shown an increased size of the PSD occurs as a result of disuse of a synapse (Murthy et al., 2001). Hypertrophy of the PSD has been also described in white deaf cats and in shaker-2 knockout mice (Redd et al., 2000; Lee et al., 2003). One explanation for this increase in the PSD size is a recruitment of new proteins to the PSD rather than the rearrangement of particles on the membrane (Gulley et al., 1977). In other systems, the expression of GluRs at the PSD increased as result of synapse disuse (Rao and Craig, 1997; Lissin et al., 1998; O’Brien et al., 1998; Turrigiano et al., 1998). Alternatively, a thicker PSD might indicate degenerative changes, as evidenced by an overall loosening of the normally compact structure (Marton et al., 1999). Data presented in this study showed that the PSD was thicker, but after 2 days of denervation its cytoplasmic face looked less condensed and at 7 days after denervation the PSD electrondense material of the PSD was not observed. The signal that initiates these responses has not been identified, although it has been suggested that the presynaptic element may play a role (Gulley et al., 1977). These data suggest that soon after cochlear ablation, there is an early phase where there is recruitment of intracellular proteins, including specific subunits of GluRs (see below) to the

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PSD to compensate the change in glutamate released from the AN. This phase might be followed by a late phase, where the PSD loses its structural and molecular components in response to deafferentation. Thus, the data described in this study indicate that the presynaptic neuron controls the structural integrity of the excitatory synapse. 4.2. Deafferentation reapportions AMPAR subunits at the AN synapse on FC After cochlear ablation, the communication between the sensory organ and the brain is altered. The results from this study showed that under these circumstances, neurons in the brainstem are capable of modulating their synaptic strength by regulating the number of GluRs at the synapse. FC responded to the loss of AN synaptic input by reapportionment the GluR2/3 and GluR4 AMPA receptor subunits at the AN synapse. Therefore, what would the effect be on FC function if the density of particular AMPAR subunits changed? Changes in the subunit composition of AMPAR at the synapse can affect the neuronal synaptic responses (Liu and Cull-Candy, 2000). However, it has not been determined how immediate changes in GluR expression in the auditory pathway affect neuronal responses after loss of sensory input. We know that the fastest receptors, those containing the AMPA GluR4 subunit, have been found in the FC in a very selective and targeted manner (Rubio and Wenthold, 1997; Gardner et al., 1999, 2001). A decrease in the expression of GluR4 at the AN synapse after peripheral injury might indicate that the actual AMPAR complex deactivate more slowly (Mosbacher et al., 1994). This circumstance might lead to slightly longer synaptic responses. Because FCs are part of cochlear nucleus neurons that initiate the ascending auditory pathways any change in their neuronal responses may affect those of downstream cells (Mossop et al., 2000). Additional studies are needed to test it, specifically, to determine whether the GluR4 flop-splice variant decreases in response to deafferentation. Additionally, data presented in this study showed that there was an upregulation of the GluR2 subunit at the AN/FC synapse as an early effect of deafferentation. The presence of a GluR2 subunit will render the channel impermeable to calcium (Geiger et al., 1995). An increase in the Ca2++ levels has been described in CN neurons after cochlear ablation (Zirpel et al., 2000). It is possible that this early increase of Ca2+ impermeable AMPA receptor complexes at the AN/FC synapse after deafferentation is important to prevent an excess of Ca2+ entry through AMPA receptors (Zirpel et al., 2000), therefore avoiding excitotoxicity and neuronal cell death. It would be of great interest to determine whether principal neurons in the ventral cochlear nucleus which contain very low levels of GluR2 (Wang et al., 1998) and therefore are more permeable to Ca2+ (Gardner et al., 2001), respond to denervation by upregulating the GluR2 subunit at the AN synapse.

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4.3. Deafferentation affects differentially the composition of AMPA receptor subunits at the excitatory synapse of PF on FC and CwC cells The PSDs of neurons associated with the same source of input may contain different receptor composition at the synapse and they might respond differentially to changes in the synaptic input. To test this hypothesis, I investigated the response of the synapses formed by the PF on FC and on CwC cells in the DCN ipsilateral to the cochlear ablation. This study presented evidence that PF synapses on apical dendrites of FC and CwC responded to AN denervation by redistributing the synaptic GluR2 and GluR3 subunits at 2 days after deafferentation. Interestingly, this redistribution differed between the two synapses. Data showed that only the synapse of PF on CwC upregulated the GluR2 subunit at the PSD, implying an increase in the number of Ca2+ impermeable AMPA receptor complexes at that particular synapse. On the other hand, the expression of GluR2 at the synapse formed by the PF on FC was found unaltered. Therefore, insertion of new GluR3 subunits at the PF/FC synapse might explain the upregulation obtained when an antibody recognizing the GluR2 and GluR3 subunits was used. Upregulation of the GluR3 at the PF/FC synapses may enable cells to express more Ca2+ permeable AMPA receptors at times of enhanced plasticity as it happens during development (Caicedo and Eybalin, 1999). This may be needed to compensate the loss of AN synaptic input after cochlear ablation. On the other hand, the insertion of more GluR3 subunits might result in the formation of different AMPA receptor complexes at the PF/FC synapses (Petralia et al., 2004). As discussed above, the number and type of receptors at the postsynaptic membrane determine the response to the neurotransmitter released from the presynaptic terminal. Therefore, an enhanced Ca2+ permeability, together with a change in the response of the PF/FC synapse upon the increase of GluR3 subunit at this synapse, may affect the electrophysiological properties of FC, and consequently their target in upper auditory nuclei. Evidence showed an enhanced excitability in the DCN projection neurons in response to cochlear trauma (Mossop et al., 2000). A possible explanation for this hyperactivity is the loss of inhibitory transmitters caused by ablation (Suneja et al., 1998a,b). An upregulation of GluRs at the synapse in response to deafferentation may also contribute to the persistence of hyperactivity in the DCN and upper targets of the auditory pathway. 4.4. Contralateral glutamatergic synapses respond to deafferentation by increasing faster AMPA receptor subunits at the synapse Data presented in this study showed that the expression of AMPAR subunits in DCN neurons is different for the separate sides of the cochlear trauma and from

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normal hearing littermates. Analysis of the glutamatergic synapses of the AN and PF on FC and/or CwCs in DCN contralateral side to cochlear ablation showed that these neurons maintain unaltered the expression of GluR2 subunit and that they probably upregulated the GluR3 at the synapse. Interestingly, this redistribution was similar for the three types of synapses independent of the cell type. The synapse of the AN on FC showed no change in the expression of GluR4. The result of the incorporation of more GluR3 subunits can result in the formation of more Ca2+ permeable channels, and consequently might enhance the ability of the synapse to exhibit the plasticity required to compensate the absence of auditory input of one side. The CN complex gives rise to widespread projections to nuclei throughout the brainstem, including the contralateral CN (Cant and Benson, 2003). Data presented in this paper showed that unilateral deafferentation also affected the contralateral CN. The imbalance of activity in CN neurons of the contralateral side may cause a change in the expression of excitatory receptors at the synapse. Contralateral excitatory inputs to the CN may compensate rapidly for large changes in afferent input (Sumner et al., 2005). Alternatively, descending excitatory projections that receive imbalance excitation from the lesion side may also contribute to a change in the expression of AMPA receptors at the synapse. More studies are needed to determine the significance and the source of the redistribution of AMPA receptors at the synapse in response to contralateral deprivation of synaptic input. Acknowledgments I thank Laurence Trussell for carefully reading the manuscript and very helpful suggestions. I am thankful to Robert Wenthold for kindly providing the antibodies for GluR2/3 and GluR4 and Andrew Moiseff for his help and expertise with the ABR test and for reading the manuscript. I thank Christina Alevras for the blinded studies of the average thickness of the PSDs. This study was supported by the University of Connecticut. I acknowledge NSF DBI-0420580 for funds to purchase the Tecnai 12 Biotwin. References Bear, M.F., Malenka, R.C., 1994. Synaptic plasticity: LTP and LTD. Curr. Opin. Neurobiol. 4, 389–399. Berrebi, A., Mugnaini, E., 1991. Distribution and targets of cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig. Anat. Embryol. 183, 427–454. Bilak, M., Kim, J., Potashner, S.J., Bohne, B.A., Morest, D.K., 1997. New growth of axons in the cochlear nucleus of adult chinchillas after acoustic trauma. Exp. Neurol. 147, 256–268. Bledsoe Jr., S.C., Nagase, S., Miller, J.M., Altschuler, R.A., 1995. Deafness-induced plasticity in the mature central auditory system. Neuroreport 7, 225–229. Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: longterm potentiation in the hippocampus. Nature 361, 31–39.

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