|Selective loss of AMPA receptor subunits at inhibitory neuron synapses in the cerebellum of the ataxic stargazer mouse.|
Olga Shevtsova,Beulah Leitch
AMPA receptor subunits (GluA1-4) are trafficked to membrane synaptic sites by transmembrane AMPA receptor regulatory proteins (TARPs). In the stargazer mutant mouse, expression of TARP-?2 (stargazin) is severely reduced, resulting in cerebellar ataxia. Stargazer granule cells (GCs) have a complete loss of functional AMPARs, as ?2 is their main TARP; hence mossy fiber (MF)-GC synapses are silent. The aim of the current study was to investigate how the stargazin deficit affects expression levels of AMPAR subunits at output synapses from GC parallel fibers (PF) onto inhibitory neurons in the molecular layer. Cerebella from male litter-pairs of stargazer and control mice were analyzed by post-embedding immunogold-microscopy. Levels of GluA2/3 and GluA4 were evaluated by measuring relative density of immunogold at PF-Purkinje cell (PF-PC) and PF-interneuron (PF-In) synapses respectively. In total, 100 synapses were analyzed in each pair of stargazer and control littermates. GluA2/3 and GluA4 expression was significantly reduced throughout the stargazer cerebellar cortex. GluA2/3 levels were reduced by 52% (p<0.001) at PF-PC synapses, and GluA4 levels by 31% (p<0.001) at PF-In synapses in stargazers. In neither case, however, was there a total loss of synaptic AMPAR subunits as occurs at MF-GC synapses. As the inhibitory neurons of the molecular layer express other TARPs in addition to stargazin, TARP compensation may limit the loss of GluA subunits at these synapses and explain why they are not silent like the MF-GC synapses. These data suggest that the ataxic phenotype in stargazers is primarily due to absence of AMPARs at cerebellar MF-GC synapses.
|Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea.|
M J Drescher,D G Drescher,K M Khan,J S Hatfield,N A Ramakrishnan,M D Abu-Hamdan,L A Lemonnier
Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine beta-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen's cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.