Gap43 transcription modulation in the adult brain depends on sensory activity and synaptic cooperation.

Brain development and learning is accompanied by morphological and molecular changes in neurons. The growth associated protein 43 (Gap43), indicator of neurite elongation and synapse formation, is highly expressed during early stages of development. Upon maturation of the brain, Gap43 is down-regulated by most neurons with the exception of subdivisions such as the CA3 region of hippocampus, the lateral superior olive (LSO) and the central inferior colliculus (CIC). Little is known about the regulation of this mRNA in adult brains. We found that the expression of Gap43 mRNA in specific neurons can be modulated by changing sensory activity of the adult brain. Using the central auditory system of rats as a model, Gap43 protein and mRNA levels were determined in LSO and CIC of hearing-experienced rats unilaterally or bilaterally deafened or unilaterally stimulated by a cochlear implant (CI). Our data indicate that Gap43 is a marker useful beyond monitoring neuronal growth and synaptogenesis, reflecting also specific patterns of synaptic activities on specific neurons. Thus, unilateral loss of input to an adult auditory system directly causes asymmetrical expression of Gap43 mRNA between LSOs or CICs on both sides of the brainstem. This consequence can be prevented by simple-patterned stimulation of a dysfunctional ear by way of a CI. We suggest that as a function of input balance and activity pattern, Gap43 mRNA expression changes as cells associate converging afferent signals

Experimental Design.

<p>T_i: time of implantation of a passive electrode dummy or onset of electrical intracochlear stimulation (EIS); d: day(s).</p

Gap43 mRNA and protein expression in auditory brainstem.

<p>(A) Anteroventral cochlear nucleus (AVCN, dashed line) was devoid of Gap43 mRNA staining on both sides under any experimental condition. (B) Faintly stained Gap43 protein-positive axonal boutons were present throughout AVCN in controls and all experimental conditions. Inset: higher magnification of immuno-positive presynaptic endings (arrowheads). (C) Gap43 protein expression in lateral olivocochlear neurons (arrows) within LSOi (dashed line) required at least 5 days (d) of electrode implantation independent of its activation. Inset: close-up of Gap43 protein-positive neurons (arrows) and boutons following 7d of <i>ud</i>. (D) Throughout CIC (dashed line), Gap43 immunoreactivity was always present. Inset: close-up of immuno-positive presynaptic endings (arrowheads). Scale bars for A to D: 0.2 mm. Scale bars of insets B to D: 20 μm. LSO: lateral superior olive; CIC: central inferior colliculus; nVIII: 8th cranial nerve; tb: trapezoid body; i: ipsilateral; c: contralateral; <i>ud</i>: unilateral deafness.</p

Expression level of Gap43 mRNA depends on synaptic cooperation in auditory brainstem nuclei.

<p>(A) Under control conditions, neurons in LSO and CIC of both sides of the brainstem receive stimulation-dependent excitatory (green) and inhibitory (red) inputs defining a functional balance with respect to metabotropic receptor activation to generate a basal level of Gap43 mRNA expression (light purple dots). (B) Induction of bilateral deafness silences all stimulation-dependent inputs (gray), leaving Gap43 mRNA levels unchanged. Question marks indicate a possible influence of spontaneous activity in MNTB neurons of unknown significance. (C) Unilateral deafness causes an imbalance of excitation and inhibition on neurons. Loss of excitation (gray) remained ineffective in modulating Gap43 mRNA levels in LSOi and CICc, whereas loss of stimulation-dependent inhibition for LSO via MNTB induces a significant increase of Gap43 mRNA staining level (dark purple dots) in LSOc and CICi. (D) Following unilateral EIS, excitatory afferents of neurons in LSOi and CICc as well as inhibitory afferents on neurons in LSOc and CICi were kept active even if the CI induced input is stronger-than-normal (thick lines). This resulted in a deviation from the normal excitatory-to-inhibitory input ratio and led to a rise of <i>gap43</i> transcription in neurons of these auditory regions (dark purple dots). nVII: 8th cranial nerve; MNTB: medial nucleus of the trapezoid body; LSO: lateral superior olive; CI: cochlear implant; CIC: central inferior colliculus; EIS: electrical intracochlear stimulation; VCN: ventral cochlear nucleus.</p

Gap43 mRNA staining in lateral superior olive (LSO).

<p>(A, B) A basal level of Gap43 mRNA was seen in neuronal cell bodies (purple dots) of the entire left (A) and right (B) LSO (dashed outline) of untreated control rats (Co). The insets show lack of staining in corresponding sections after use of Gap43 sense probe. Scale bar for insets: 0.2 mm. (C, D) Gap43 mRNA expression in left (C) and right (D) LSO after 5 days (d) of bilaterally deaf (<i>bd</i>) rats is equivalent to control level. Inset in D shows Gap43 mRNA positive neurons at higher magnification. Scale bar of inset: 20 μm. (E) Following 3d of unilateral deafness (<i>ud</i>), Gap43 mRNA expression decreased in neurons of LSOi compared to controls. (F) Simultaneously, the expression increased contralaterally. (G, H) After 5d of unilateral stimulation (<i>us</i>), high bilaterally balanced Gap43 mRNA levels were observed. Scale bar for A to H: 0.2 mm. SPON: superior paraolivary nucleus; MSO: medial superior olive; l: left; r: right; i: ipsilateral; c: contralateral.</p

The effectiveness of electrical intracochlear stimulation (EIS) on neurons of the anteroventral cochlear nucleus (AVCN).

<p>Fos mRNA (A) and Fos protein (B) closely corresponded locally after EIS for under 2 h duration. (A, C) Fos mRNA expression (black dots) in neurons of ipsilateral (i) AVCN after 73 min (A; cp. Illing and Rosskothen-Kuhl, 2012) and 1 day (d) (C) of unilateral stimulation <i>(us</i>). Fos-positive neurons were spatially limited to regions tonotopically corresponding to frequencies processed at the site of intracochlear electrode position. The number of labeled neurons was higher after 73 min than after 1d of <i>us</i>. Inset A: AVCNi was devoid of staining after incubation with Fos sense probe. Inset C: higher magnification of mRNA-positive neurons, showing strongest staining in cytoplasm. (B, D) Fos protein staining (black dots) in AVCNi after 73 min (B; cp. Illing and Rosskothen-Kuhl, 2012) and 1d (D) of <i>us</i>. Protein expression was spatially limited to a band tonotopically corresponding to intracochlear stimulation position. Inset B: AVCNi of control. Scale bars in insets A and B: 0.2 mm. Inset D: higher magnification of Fos protein positive nuclei in AVCNi. Scale bars in insets C and D: 20 μm. (E) Three days after sustained EIS a strong decrease in the number of Fos protein positive nuclei was observable. (F) Following 5d of <i>us,</i> no further protein positive nuclei were detectable. Scale bar for A to E: 0.2 mm. nVIII: 8th cranial nerve.</p

Quantification of Gap43 mRNA staining intensities in LSO (A, B) and CIC (C, D).

<p>Bilateral comparison of left-to-right or ipsilateral-to-contralateral staining intensities in LSO (A) and CIC (C) indicates that Gap43 mRNA was different between both sides of the brainstem if sensory stimulation was reduced on one side only (LSO: n = 19/92 rats/slices; F = 45.48, DFn = 2, DFd = 118; <i>ud</i>: 0.39±0.02; Co: 0.96±0.03; CIC: n = 19/89 rats/slices; F = 201.5, DFn = 2, DFd = 116; <i>ud</i>: 1.82±0.08; Co: 1.03±0.03; p<0.001 for both). EIS in the ear of one side with the other ear continuing transduction of acoustically signals maintained ipsilateral-to-contralateral balance as in controls (LSO: n = 6/29 rats/slices; <i>us</i>: 1.03±0.03; CIC: n = 6/30 rats/slices, <i>us</i>: 1.06±0.03; p>0.05 for both). Dotted line indicates bilateral symmetry (1.0). Significant differences against control level are indicated by asterisks above bars. Significant divergences between <i>ud</i> and <i>us</i> rats are indicated by lines with associated asterisks. (B) Staining results of <i>gap43</i> transcription in LSO (n = 29/142 rats/slices; F = 53.89, DFn = 5, DFd = 278) indicates that the staining intensity increased significantly against controls (Co) in LSOc due to unilateral deafness (<i>ud</i>; n = 19/89 rats/slices; p<0.001), and in bilateral LSO after unilateral stimulation (<i>us</i>; n = 14/55 rats/slices; p<0.001 for both). Additionally, staining intensity in <i>ud</i> rats on the contralateral side was significantly higher than for bilaterally deafened (<i>bd</i>) rats (n = 15/87 rats/slices; ###: p<0.001). Staining intensities of both LSOs of <i>us</i> rats were higher than the ipsilateral intensity of <i>ud</i> rats (n = 17/90 rats/slices; p<0.001 for both). (D) Staining results of <i>gap43</i> transcription in CIC (n = 29/145 rats/slices; F = 18.71, DFn = 5, DFd = 284) revealed that the level increased against controls in CICi due to <i>ud</i> (n = 19/91 rats/slices; p<0.001), and in bilateral CIC after <i>us</i> (n = 14/56 rats/slices; p = 0.0085 for Co vs. <i>us</i> i; p = 0.0385 for Co vs. <i>us</i> c). Mean staining intensity of CIC of <i>bd</i> rats was significantly different from CICi of <i>ud</i> rats (n = 15/89 rats/slices; ###: p<0.001), whereas both sides of <i>us</i> rats rose against CICc of <i>ud</i> rats (n = 17/95 rats/slices; p = 0.0007 for <i>us</i> i vs. <i>ud</i> c; p = 0.0049 for <i>us</i> c vs. <i>ud</i> c). Significance levels: (***/^###) for p<0.001, (**) for p<0.01, (*) for p<0.05. LSO: lateral superior olive; CIC: central inferior colliculus; i: ipsilateral; c: contralateral.</p

Experimental Groups.

<p>c: contralateral; i: ipsilateral; Co: control; <i>bd</i>: bilateral deaf; <i>ud</i>: unilateral deaf; <i>us:</i> unilateral stimulation; EIS: electrical intracochlear stimulation.</p

Gap43 mRNA staining in central inferior colliculus (CIC).

<p>(A, B) In the untreated control group (Co), Gap43 mRNA positive neurons (purple dots) in CIC (dashed outline) were mainly localized ventrally (arrows). (C, D) 3 days (d) after unilateral deafness <i>(ud</i>), Gap43 mRNA level increased in CICi (C), while expression was decreased in CICc (D). The inset shows a higher magnification of stained CIC neurons. Scale bar in inset: 20 μm. (E, F) After 70d of <i>ud</i>, Gap43 mRNA expression rose bilaterally in CIC, with a significantly higher level on the ipsilateral side (E) compared to the contralateral side (F). Insets show control staining after incubation with Gap43 sense probe. Scale bar for insets: 0.2 mm. (G, H) Unilateral stimulation (<i>us</i>) for 7d resulted in an increase of Gap43 mRNA levels in ventral CIC on ipsilateral (G) and contralateral (H) side. Scale bar for A to H: 0.2 mm. l: left; r: right; i: ipsilateral; c: contralateral; CG: central gray.</p