Disynaptic Inhibitory Cerebellar Control Over Caudal Medial Accessory Olive.

The olivocerebellar system, which is critical for sensorimotor performance and learning, functions through modules with feedback loops. The main feedback to the inferior olive comes from the cerebellar nuclei (CN), which are predominantly GABAergic and contralateral. However, for the subnucleus d of the caudomedial accessory olive (cdMAO), a crucial region for oculomotor and upper body movements, the source of GABAergic input has yet to be identified. Here, we demonstrate the existence of a disynaptic inhibitory projection from the medial CN (MCN) to the cdMAO via the superior colliculus (SC) by exploiting retrograde, anterograde, and transsynaptic viral tracing at the light microscopic level as well as anterograde classical and viral tracing combined with immunocytochemistry at the electron microscopic level. Retrograde tracing in Gad2-Cre mice reveals that the cdMAO receives GABAergic input from the contralateral SC. Anterograde transsynaptic tracing uncovered that the SC neurons receiving input from the contralateral MCN provide predominantly inhibitory projections to contralateral cdMAO, ipsilateral to the MCN. Following ultrastructural analysis of the monosynaptic projection about half of the SC terminals within the contralateral cdMAO are GABAergic. The disynaptic GABAergic projection from the MCN to the ipsilateral cdMAO mirrors that of the monosynaptic excitatory projection from the MCN to the contralateral cdMAO. Thus, while completing the map of inhibitory inputs to the olivary subnuclei, we established that the MCN inhibits the cdMAO via the contralateral SC, highlighting a potential push-pull mechanism in directional gaze control that appears unique in terms of laterality and polarity among olivocerebellar modules.

Morphofunctional alterations in the olivocochlear efferent system of the genetic audiogenic seizure-prone hamster GASH:Sal.

The genetic audiogenic seizure hamster (GASH:Sal) is a model of a form of reflex epilepsy that is manifested as generalized tonic-clonic seizures induced by external acoustic stimulation. The morphofunctional alterations in the auditory system of the GASH:Sal that may contribute to seizure susceptibility have not been thoroughly determined. In this study, we analyzed the olivocochlear efferent system of the GASH:Sal from the organ of Corti, including outer and inner hair cells, to the olivocochlear neurons, including shell, lateral, and medial olivocochlear (LOC and MOC) neurons that innervate the cochlear receptor. To achieve this, we carried out a multi-technical approach that combined auditory hearing screenings, scanning electron microscopy, morphometric analysis of labeled LOC and MOC neurons after unilateral Fluoro-Gold injections into the cochlea, and 3D reconstruction of the lateral superior olive (LSO). Our results showed that the GASH:Sal exhibited higher auditory brain response (ABR) thresholds than their controls, as well as absence of distortion-product of otoacoustic emissions (DPOAEs) in a wide range of frequencies. The ABR and DPOAE results also showed differences between the left and right ears, indicating asymmetrical hearing alterations in the GASH:Sal. These alterations in the peripheral auditory activity correlated with morphological alterations. At the cochlear level, the scanning electron microscopy analysis showed marked distortions of the stereocilia from basal to apical cochlear turns in the GASH:Sal, which were not observed in the control hamsters. At the brainstem level, MOC, LOC, and shell neurons had reduced soma areas compared with control animals. This LOC neuron shrinkage contributed to reduction in the LSO volume of the GASH:Sal as shown in the 3D reconstruction analysis. Our study demonstrated that the morphofunctional alterations of the olivocochlear efferent system are innate components of the GASH:Sal, which might contribute to their susceptibility to audiogenic seizures. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Descending projections from the inferior colliculus to medial olivocochlear efferents: Mice with normal hearing, early onset hearing loss, and congenital deafness.

Auditory efferent neurons reside in the brain and innervate the sensory hair cells of the cochlea to modulate incoming acoustic signals. Two groups of efferents have been described in mouse and this report will focus on the medial olivocochlear (MOC) system. Electrophysiological data suggest the MOC efferents function in selective listening by differentially attenuating auditory nerve fiber activity in quiet and noisy conditions. Because speech understanding in noise is impaired in age-related hearing loss, we asked whether pathologic changes in input to MOC neurons from higher centers could be involved. The present study investigated the anatomical nature of descending projections from the inferior colliculus (IC) to MOCs in 3-month old mice with normal hearing, and in 6-month old mice with normal hearing (CBA/CaH), early onset progressive hearing loss (DBA/2), and congenital deafness (homozygous Shaker-2). Anterograde tracers were injected into the IC and retrograde tracers into the cochlea. Electron microscopic analysis of double-labelled tissue confirmed direct synaptic contact from the IC onto MOCs in all cohorts. These labelled terminals are indicative of excitatory neurotransmission because they contain round synaptic vesicles, exhibit asymmetric membrane specializations, and are co-labelled with antibodies against VGlut2, a glutamate transporter. 3D reconstructions of the terminal fields indicate that in normal hearing mice, descending projections from the IC are arranged tonotopically with low frequencies projecting laterally and progressively higher frequencies projecting more medially. Along the mediolateral axis, the projections of DBA/2 mice with acquired high frequency hearing loss were shifted medially towards expected higher frequency projecting regions. Shaker-2 mice with congenital deafness had a much broader spatial projection, revealing abnormalities in the topography of connections. These data suggest that loss in precision of IC directed MOC activation could contribute to impaired signal detection in noise.

Adenomatous Polyposis Coli Protein Deletion in Efferent Olivocochlear Neurons Perturbs Afferent Synaptic Maturation and Reduces the Dynamic Range of Hearing.

Normal hearing requires proper differentiation of afferent ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) that carry acoustic information to the brain. Within individual IHCs, presynaptic ribbons show a size gradient with larger ribbons on the modiolar face and smaller ribbons on the pillar face. This structural gradient is associated with a gradient of spontaneous rates and threshold sensitivity, which is essential for a wide dynamic range of hearing. Despite their importance for hearing, mechanisms that direct ribbon differentiation are poorly defined. We recently identified adenomatous polyposis coli protein (APC) as a key regulator of interneuronal synapse maturation. Here, we show that APC is required for ribbon size heterogeneity and normal cochlear function. Compared with wild-type littermates, APC conditional knock-out (cKO) mice exhibit decreased auditory brainstem responses. The IHC ribbon size gradient is also perturbed. Whereas the normal-developing IHCs display ribbon size gradients before hearing onset, ribbon sizes are aberrant in APC cKOs from neonatal ages on. Reporter expression studies show that the CaMKII-Cre used to delete the floxed APC gene is present in efferent olivocochlear (OC) neurons, not IHCs or SGNs. APC loss led to increased volumes and numbers of OC inhibitory dopaminergic boutons on neonatal SGN fibers. Our findings identify APC in efferent OC neurons as essential for regulating ribbon heterogeneity, dopaminergic terminal differentiation, and cochlear sensitivity. This APC effect on auditory epithelial cell synapses resembles interneuronal and nerve-muscle synapses, thereby defining a global role for APC in synaptic maturation in diverse cell types. SIGNIFICANCE STATEMENT: This study identifies novel molecules and cellular interactions that are essential for the proper maturation of afferent ribbon synapses in sensory cells of the inner ear, and for normal hearing.

A direct hippocampo-cerebellar projection in chicken.

Our previous studies suggested that a direct hippocampo-cerebellar projection might exist in the chicken. To confirm such a presumption of hippocampo-cerebellar interactions, horseradish peroxidase (HRP) was used as a retrograde tracer to be injected into the white matter of the folia VI-VIII of the cerebellum in young and adult chickens. In another set of experiments, young chickens were subjected to electrolytic lesions of the hippocampal formation (HF), especially the ventromedial portion, and the cerebellum was observed with the electron microscope to find neuronal degeneration in the HF. Following injections of HRP into the cerebellum, a large number of labeled neurons were found in the area APHm-APHim of the HF in the young and adult chickens. As a result of the electrolytic lesions of the APHm-APHim in the HF, many large degenerated nerve fibers were found in the white matter in the vicinity of the lateral nucleus of the cerebellum, and some small degenerated fibers were found in the white matter of the folia VI-VIII. In the cerebellar cortex of folia VI-VIII, degenerated axonal terminals occurred in both the molecular and Purkinje layers, but not in the granular layer. In the lateral nucleus, some dark degenerating axonal terminals were recognized to connect with the perikarya of neurons of this nucleus. The present experiments demonstrate that the APHm-APHim of the HF directly projected to the cortex of folia VI-VIII and the lateral nucleus of the cerebellum in young chickens.

CD8(+)/perforin/granzyme B(+) effector cells infiltrating cerebellum and inferior olives in gluten ataxia.

Up to 8% of patients with gluten sensitivity (GS) develop neurological symptoms such as ataxia, dementia, seizures or peripheral neuropathy. The underlying immunological mechanisms still remain to be elucidated. We here report the case of a 68-year-old male patient suffering from progressive ataxia and dementia associated with chronic diarrhea and both elevated IgG and IgA antigliadin-antibodies. At autopsy, frequent argyrophilic glial and neuronal inclusions within the basal nucleus of Meynert were considered as the structural correlative for the cognitive decline. Significant neuronal loss in the cerebellar cortex and the inferior olives was accompanied by infiltrating CD8(+)/perforin(+)/granzyme B(+) cells as well as reactive astrogliosis and microglial activation. These CD8(+) cytotoxic T and NK cells are likely to act as effector cells responsible for neuronal cell death in patients with gluten sensitivity and neurological disease and might therefore at least partly be responsible for cerebellar symptoms in gluten ataxia. In conclusion, our results, showing an absence of B- or plasma cells but multiple CD8(+) as well as granzyme B and perforin expressing cells in ataxia-associated brain areas, suggest that there are also prominent cytotoxic effects in neuropathogenesis of GS.

Normal adult climbing fiber monoinnervation of cerebellar Purkinje cells in mice lacking MHC class I molecules.

Some immune system proteins have recently been implicated in the development and plasticity of neuronal connections. Notably, proteins of the major histocompatibility complex 1 (MHC class 1) have been shown to be involved in synaptic plasticity in the hippocampus and the development of projection patterns in the visual system. We examined the possible role for the MHC class 1 proteins in one well-characterized example of synaptic exuberance and subsequent refinement, the climbing fiber (CF) to Purkinje cell (PC) synapse. Cerebella from adult mice deficient for two MHC genes, H2-D1 and H2-K1, and for beta2-microglobulin gene were examined for evidence of deficient elimination of supernumerary CF synapses on their PCs. Electrophysiological and morphological evidence showed that, despite the absence of these MHC class 1 molecules, adult PCs in these transgenic mice are monoinnervated as in wild-type animals. These findings indicate that, at the level of restriction of afferent number at this synapse, functional MHC class 1 proteins are not required.

Neuroglia in the inferior olivary nucleus during normal aging and Alzheimer's disease.

It is likely that neuronal loss occurs in certain brain regions in Alzheimer's Disease (AD) without any neurofibrillary pathology. In the human principle inferior olivary nucleus (PO), we have shown that neuronal loss is about 34% (Lasn et al. Journal of Alzheimer Disease, 2001; 3: 159-168), but the fate of the neuroglial cells is unknown. Since the unique network of neurons and neuroglial cells and their cohabitation are essential for normal functioning of CNS, we designed a study to estimate the total number of oligodendrocytes and astrocytes in normally aged and AD brains. The study is based on 10 control and 11 AD post-mortem human brains. An unbiased stereological fractionator method was used. We found significant oligodendroglial cell loss (46%) in AD as compared to control brains, while the total number of astrocytes showed a tendency to decrease. It is likely that the ratio of oligodendroglial cells to neurons remains unchanged even in degenerative states, indicating that oligodendroglial cells parallel neuronal loss. Astroglial cells did not increase in total number, but the ratio to neurons was significantly increased due to the neuronal loss. Using a novel unbiased quantitative method, we were able to describe significant oligodendroglial loss in the PO but the pathogenic mechanism behind remains unknown.

Spatiotemporal distribution of Connexin45 in the olivocerebellar system.

The olivocerebellar system is involved in the transmission of information to maintain sensory motor coordination. Gap junctions have been described in various types of neurons in this system, including the neurons in the inferior olive that provide the climbing fibers to Purkinje cells. While it is well established that Connexin36 is necessary for the formation of these neuronal gap junctions, it is not clear whether these electrical synapses can develop without Connexin45. Here we describe the development and spatiotemporal distribution of Connexin45 in relation to that of Connexin36 in the olivocerebellar system. During development Connexin45 is expressed in virtually all neurons of the inferior olive and cerebellar nuclei. During later postnatal development and adulthood there is a considerable overlap of expression of both connexins in subpopulations of all main olivary nuclei and cerebellar nuclei as well as in the stellate cells in the cerebellar cortex. Despite this prominent expression of Connexin45, ultrastructural analysis of neuronal gap junctions in null-mutants of Connexin45 showed that their formation appears normal in contrast to that in knockouts of Connexin36. These morphological data suggest that Connexin45 may play a modifying role in widely distributed, coupled neurons of the olivocerebellar system, but that it is not essential for the creation of its neuronal gap junctions.

Purkinje cell spinogenesis during architectural rewiring in the mature cerebellum.

Spines can grow and retract within hours of activity perturbation. We investigated the time course of spine formation in a model of plasticity involving changes in brain architecture where spines of a dendritic domain become innervated by a different neuronal population. Following a lesion of rat olivocerebellar axons, by severing the inferior cerebellar peduncle, new spines grow on the deafferented proximal dendrite of the Purkinje cells (PCs) and these new spines become innervated by parallel fibres (PFs) that normally contact only the distal dendrites. The varicosities of climbing fibre (CF) terminal arbors disappear within 3 days of the lesion. Spine density in the proximal dendritic domain begins to rise within 3 days and continues to increase towards a plateau at 6-8 days. In 'slow Wallerian degeneration' mice, in which axonal degeneration is delayed, climbing fibre varicosities virtually disappear at 14 rather than 3 days. Spine density in the proximal dendritic domain is similar to control Purkinje cells up to 14 days and increases significantly 18 days postlesion. The delayed spinogenesis in the latter mutant is the result of a persistence of the climbing fibre presynaptic structure in the absence of activity. Therefore, climbing fibre activity itself is not directly responsible for the suppression of spine formation, but suppression mechanisms tend to become weaker as long as the structural dismantling of the presynaptic varicosities proceeds. Thus, spinogenesis is guided by two different mechanisms; a rapid one related to changes in homotypic remodeling and a slower one, which requires the removal of a competitive afferent.

CNTFRalpha and CNTF expressions in the auditory brainstem: light and electron microscopy study.

CNTF receptor alpha (CNTFRalpha) is involved in the development, the maintenance and the regeneration of a variety of brain structures. However, its in vivo distribution has not been determined in the auditory system. CNTFRalpha expression was studied in developing and adult rat brainstem auditory nuclei using immunohistochemistry. At birth, the CNTFRalpha immunolabeling was clearly present in somata of the external nucleus of the inferior colliculus but was diffuse throughout brainstem auditory nuclei. The labeling was present in most brainstem auditory nuclei by post-natal day (PND) 6. The intensity of the staining subsequently increased to its highest level at PND21 and decreased to an adult-like appearance by the fourth post-natal week. In adult, CNTFRalpha labeling occurred in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial superior olive (MSO), and the medial nucleus of the trapezoid body (MNTB). CNTFRalpha labeling first appeared in the central nucleus of the inferior colliculus (IC) by the end of the fourth week. There was a general increase in the expression of CNTFRalpha that begins prior to the onset of hearing and reaches its highest level after this important developmental stage. Ultrastructural analysis in the adult ventral CN revealed the presence of CNTFR in post-synaptic sites. The presence of CNTF has been investigated in the adult using both Western blot and immunohistochemistry. Western blot showed the presence of CNTF in both peripheral and central auditory structures. The CNTF label was generally localized to the somatic compartment, in axons and as puncta surrounding neuronal cell bodies and dendrites. Differential CNTF labeling was observed between the different auditory nuclei. CNTF staining is present in neurons of the CN, the MNTB and the LSO, while it is restricted to axons and puncta surrounding neuronal somata in the IC. The clear presence of CNTFRalpha at post-synaptic terminals and that of its ligand the CNTF in axons and puncta surrounding neuronal cell bodies suggest an anterograde mode of action for CNTF in the central auditory system.

The postnatal developmental expression pattern of urocortin in the rat olivocerebellar system.

Urocortin belongs to the family of corticotropin-releasing factor (CRF)-like peptides, which play an important role in sensorimotor coordination. CRF induces locomotor activity, and urocortin has an inhibitory effect. Here, we document the regional and subcellular localization of urocortin in the developing rat cerebellum to compare it with CRF. During the first postnatal week, urocortin immunoreactivity (UCN-ir), within the white matter and cerebellar cortex, was strongest in vermal lobules I, II, IX, and X, closely followed by lobules IV, V, and VIII; lobules VI and VII showed the weakest labeling. Cortical immunoreactivity was in the form of puncta that encircled Purkinje cell somata. By postnatal day (PD) 12, UCN-ir had increased appreciably in all lobules. In Purkinje cells, labeling was spread throughout their somata and proximal dendrites. By PD 15, labeling in lobules I-IV appeared to wane, yet still prevailed in the central and posterior lobules. This anterior-to-posterior gradient persisted through to adulthood. The study shows that urocortin and CRF have similar regional distribution profiles during development, suggesting synergistic roles within the vestibulocerebellum. The onset of the adult distributional pattern of urocortin at the stage when rats are capable of fluent walking patterns further strengthens the correlation between CRF-like peptides and postural control. An important difference between urocortin and CRF is the localization of urocortin, and not CRF, within Purkinje cells, implying that urocortin probably has an additional role in modulating the signals emanating from the cerebellar cortex to the deep cerebellar nuclei.

Subtype switching of vesicular glutamate transporters at parallel fibre-Purkinje cell synapses in developing mouse cerebellum.

Two subtypes of the vesicular glutamate transporter are expressed differentially in two excitatory afferents synapsing on to Purkinje cells: VGluT1 (BNPI) in axon terminals of cerebellar granule cells (i.e. parallel fibres; PFs) and VGluT2 (DNPI) in those of the inferior olivary neurons (climbing fibres; CFs). In the present study, we examined their expression in the developing mouse cerebellum. By in situ hybridization, the inferior olivary nucleus selectively expressed VGluT2 mRNA through postnatal life. In the cerebellum, both subtypes were transcribed in the external and internal granular layers during the first postnatal week. Thereafter, VGluT1 mRNA showed marked upregulation in the internal granular layer, whereas VGluT2 mRNA disappeared from the external and internal granular layers by the end of the third postnatal week. By immunohistochemistry, CF terminals consistently exhibited VGluT2 immunoreactivity in the postnatal cerebellum. By contrast, in the first 10 days of postnatal life, VGluT2 predominated in PF terminals, despite the transcription of both transporters in developing granule cells. During the second 10 days, VGluT2 in PF terminals was replaced with VGluT1 from deep regions of the molecular layer upwards, correlating with dendritic translocation of CFs. This replacement was accomplished by postnatal day 30. Taking that late-borne PFs are laid down successively on earlier ones in the molecular layer, the deep-to-superficial replacement represents maturation-linked switching from VGluT2 to VGluT1 in individual PFs, and is likely to be regulated at both the transcription and translation levels.

Sub-cellular pathology of scrapie: coated pits are increased in PrP codon 136 alanine homozygous scrapie-affected sheep.

Sub-cellular studies of transmissible spongiform encephalopathies (TSEs) have been carried out on several animal species and human beings. However, studies of optimal perfusion-fixed tissues have largely been confined to examination of rodents. Using a recently developed technique, heads of scrapie-affected sheep and controls were perfusion fixed with mixed aldehydes. The obexes were immunohistochemically labelled with PrP antibodies, and the dorsal motor nucleus of the vagal nerve was examined by electron microscopy. Irregular neuritic profiles with highly invaginated membranes, associated with coated pits were found in all scrapie-affected sheep, but not in controls. Interestingly, they were consistently more frequent in the homozygous A(136) sheep. This is the first report describing sub-cellular differences in pathology associated with different PrP genotypes. Rarely, amorphous material, or sparse fibrillar structures, were present in the extracellular space. The changes were often associated with irregular plasmalemma and frequent coated pits. Vacuolation typical of TSEs, dystrophic neurites and variable gliosis were present. Herniation of membranes and organelles from apparently healthy processes into adjacent vacuoles and dendrites was also observed. We suggest that the increase in coated pits and plasmalemma invagination is related to an attempted internalisation of aggregated disease-specific PrP, or protofilaments, from the extracellular space.

Addition of inhibition in the olivocerebellar system and the ontogeny of a motor memory.

The developmental emergence of learning has traditionally been attributed to the maturation of single brain regions necessary for learning in adults, rather than to the maturation of synaptic interactions within neural systems. Acquisition and retention of a simple form of motor learning, classical conditioning of the eyeblink reflex, depends on the cerebellum and interconnected brainstem structures, including the inferior olive. Here, we combined unit recordings from Purkinje cells in eye regions of the cerebellar cortex and quantitative electron microscopy of the inferior olive to show that the developmental emergence of eyeblink conditioning in rats is associated with the maturation of inhibitory feedback from the cerebellum to the inferior olive. The results are consistent with previous work in adult animals and indicate that the maturation of cerebellar inhibition within the inferior olive may be a critical factor for the formation and retention of learning-specific cerebellar plasticity and eyeblink conditioning.

Ultrastructural transynaptic effects of unilateral cochlear ablation in the gerbil medial superior olive.

This study investigated the long-term effects of unilateral hearing loss on the structure of synapses within the gerbil medial superior olivary (MSO) nuclei. Five animals had complete (surgical) left cochlear ablation at postnatal day 18. Previous studies have shown this to produce, within 3 days, significant transneuronal atrophy in the left dendritic field of both MSOs. Electron micrographs from sagittal ultrathin sections through the MSOs of the cochlear-ablated animals were compared to those from unoperated normals. Qualitatively, the ultrastructural features were similar. Most of the axodendritic terminals were R-type (round-type vesicles, putative excitatory) whereas, in the central part of the nucleus, predominated by neuron soma profiles, terminals of P- and F-type (pleomorphic- and flattened-type vesicles, putative inhibitory) were present in equal numbers with R-type terminals. F-type terminals were infrequent and occurred most around lateral parts of the MSO somata. These three types of terminals seen around the somata and proximal dendrites all had extended profiles with multiple, discontinuous appositions. Quantitative analysis revealed that R-type axodendritic terminals became smaller and less densely populated with vesicles where they synapsed onto the remaining dendrites arrayed towards the ablated side of both MSOs, and axosomatic P-type afferent terminals were smaller in the contralateral nuclei. A significant reduction in the number of terminals and synapses occurred in the central, somatic, region of the ipsilateral MSO. However, the terminal vesicle concentration in the remaining terminals increased. The results indicate that cochlear ablation can induce transynaptic reduction in the size of afferent axon terminals within the MSO, and alter their vesicle concentration. These changes are likely to affect the probability of transmitter release and thus influence their signaling power within the nucleus.

Differentiation of spinous synapses in the mouse organ of corti.

The inner hair cells, the primary auditory receptors, are perceived only as a means for transfer of sound signals via the auditory nerve to the central nervous system. During initial synaptogenesis, they receive relatively few and mainly somatic synapses. However, around the onset of hearing (10-14 postnatal days in the mouse), a complex network of local spinous synapses differentiates, involving inner hair cells, their afferent dendrites, and lateral olivocochlear terminals. Inner hair cell spines participate in triadic synapses between olivocochlear terminals and afferent dendrites. Triadic synapses have not yet been confirmed in the adult. Synaptic spines of afferent dendrites form axodendritic synapses with olivocochlear terminals and somatodendritic synapses with inner hair cells. The latter are of two types: ribbon-dendritic spines and stout dendritic spines surrounded only by a crown of synaptic vesicles. Formation of spinous afferent synapses results from sprouting of dendritic filopodia that intussuscept inner hair cell cytoplasm. This process continues in the adult, indicating ongoing synaptogenesis. Spinous processes of olivocochlear synaptic terminals contact adjacent afferent dendrites, thus integrating their connectivity. They develop about 14 postnatal days, but their presence in the adult has yet to be confirmed. Differentiation of spinous synapses in the organ of Corti results in a total increase of synaptic contacts and in a complexity of synaptic arrangements and connectivity. We propose that spinous synapses provide the morphological substrate for local processing of initial auditory signals within the cochlea.

A single packet of transmitter does not saturate postsynaptic glutamate receptors.

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.

Axodendritic and dendrodendritic synapses within outer spiral bundles in a human.

Axodendritic and dendrodendritic synapses have been described at the level of the outer spiral bundle (OSB) (Nadol, J.B., Jr., 1983. Laryngoscope 93, 780-791; Bodian, D., 1978. Proc. Natl. Acad. Sci. USA 75, 4582-4586). The objectives of this study were to quantify these synaptic interactions and to describe their ultrastructural morphology in a young human subject. The temporal bone of an 8-month old infant was processed for transmission electron microscopy and semiserial section reconstructions of the three OSBs were performed. The nerve fibers ((NFs)) forming the OSBs were found to segregate into two morphological groups: (1) vesicle-rich and neurofilament-poor (VR/NP); (2) vesicle-poor and neurofilament-rich (VP/NR). Synapses between VR/NP and VP/NR NFs and synapses between two VP/NR NFs were quantified. Presumed axodendritic synapses (i.e. between VR/NP and VP/NR NFs) were numerous and their numbers decreased from the first towards the third row. Presumed dendrodendritic synapses (i.e. between two VP/NR NFs) were also frequent but their numbers did not vary significantly among different rows. The presence of axodendritic synapses may provide the morphological basis for modulation of the function of the type II spiral ganglion cells (type II's) by the olivocochlear efferent system. Similarly, numerous presumed dendrodendritic synapses may provide a morphological substrate for interaction between dendrites of type II's.

Developmental changes in the localisation of the mGluR1alpha subtype of metabotropic glutamate receptors in Purkinje cells.

The regulation of neurotransmitter receptors during synapse formation has been studied extensively at the neuromuscular junction, but little is known about the development of excitatory neurotransmitter receptors during synaptogenesis in central synapses. In this study we show qualitatively and quantitatively that a receptor undergoes changes in localisation on the surface of rat Purkinje cells during development in association with its excitatory synapses. The presence of mGluR1alpha at parallel and climbing fibre synapses on developing Purkinje cells was studied using high-resolution immunoelectron microscopy. Immunoreactivity for mGluR1alpha was detected from embryonic day 18 in Purkinje cells, and showed dramatic changes in its localisation with age. At early postnatal ages (P0 and P3), mGluR1alpha was found both in somata and stem dendrites but was not usually associated with synaptic contacts. At P7, mGluR1alpha became concentrated in somatic spines associated with climbing fibres and in the growing dendritic arborisation even before innervation by parallel fibres. During the second and third postnatal week, when spines and parallel fibre synapses were generated, mGluR1alpha became progressively concentrated in the molecular layer, particularly in the synaptic specialisations. As a result, during the fourth postnatal week, the pattern and level of mGluR1alpha expression became similar to the adult and mGluR1alpha appeared in high density in perisynaptic sites. Our results indicate that mGluR1alpha is present in the developing Purkinje cells prior to their innervation by climbing and parallel fibres and demonstrate that this receptor undergoes a dynamic and specific regulation during postnatal development in association with the establishment of synaptic inputs to Purkinje cell.