Distinct Cell Classes Research Articles

Carotid body thin slices: responses of glomus cells to hypoxia and K+-channel blockers

We describe the rat carotid body thin slice preparation, which allows to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. We observed several electrophysiologically distinct cell classes within the same glomerulus. A voltage- and Ca(2+)-dependent component of the whole cell K(+) current was reversibly inhibited by low P(O(2)) (20 mmHg). Exposure of the cells to hypoxia elicited the appearance of spike-like exocytotic events. This response to hypoxia was reversible and required extracellular Ca(2+) influx. Addition of tetraethylammonium (TEA, 2-5 mM) to the extracellular solution induced in most (>95%) cells tested a secretory response similar to that elicited by low P(O(2)). Cells non-responsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to that of hypoxia or TEA was also observed after treatment of the cells with iberiotoxin to block selectively maxi-K(+) channels. Our data further support the view that membrane ion channels are critically involved in sensory transduction in the carotid body. We demonstrate that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiate the secretory response to low P(O(2)).

Morphology of horizontal cells in the retina of the capuchin monkey, Cebus apella: How many horizontal cell classes are found in dichromatic primates?

The morphology of horizontal cells was studied in the retina of dichromatic capuchin monkeys, Cebus apella. The cells were labeled with the carbocyanine dye, 1,1′,dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the labeling was then photoconverted to a stable product by using a diaminobenzidine reaction. The sizes of cell body, dendritic field, and axon terminal, as well as the number of dendritic clusters and cone convergence, were measured at increasing distance from the fovea. Three distinct morphological classes of horizontal cells were identified. Their dendritic and axonal morphology resembles those of H1, H2, and H3 cells described in trichromatic primates. The size of the cell bodies, dendritic fields, and axon terminals of all cell classes increases towards retinal periphery. H3 cells have larger dendritic fields and more dendritic clusters than H1 cells. All labeled horizontal cells located in selected patches of retina were further analyzed to quantify the differences between H1 and H3 cells. H1 cells have smaller dendritic field area, smaller total length of primary dendrites, more dendritic branching points, and larger fractal dimension than H3 cells. We have distinguished H1 and H3 cells based solely in morphological criteria. Their physiology should be further analyzed with detail, but their presence in both dichromatic and trichromatic primates suggests that neither of them have a specialized role in the red-green color opponent channel of color vision. J. Comp. Neurol. 443:105–123, 2002. © 2002 Wiley-Liss, Inc.

Morphology of horizontal cells in the retina of the capuchin monkey, Cebus apella: How many horizontal cell classes are found in dichromatic primates?

The morphology of horizontal cells was studied in the retina of dichromatic capuchin monkeys, Cebus apella. The cells were labeled with the carbocyanine dye, 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and the labeling was then photoconverted to a stable product by using a diaminobenzidine reaction. The sizes of cell body, dendritic field, and axon terminal, as well as the number of dendritic clusters and cone convergence, were measured at increasing distance from the fovea. Three distinct morphological classes of horizontal cells were identified. Their dendritic and axonal morphology resembles those of H1, H2, and H3 cells described in trichromatic primates. The size of the cell bodies, dendritic fields, and axon terminals of all cell classes increases towards retinal periphery. H3 cells have larger dendritic fields and more dendritic clusters than H1 cells. All labeled horizontal cells located in selected patches of retina were further analyzed to quantify the differences between H1 and H3 cells. H1 cells have smaller dendritic field area, smaller total length of primary dendrites, more dendritic branching points, and larger fractal dimension than H3 cells. We have distinguished H1 and H3 cells based solely in morphological criteria. Their physiology should be further analyzed with detail, but their presence in both dichromatic and trichromatic primates suggests that neither of them have a specialized role in the red-green color opponent channel of color vision.

Pharmacological heterogeneity of γ-aminobutyric acid receptors during development suggests distinct classes of rat cerebellar granule cells in situ

The γ-aminobutyric acid receptor (GABA AR) represents a ligand-gated Cl −-channel assembling as heteropentamere from 19 known subunits. Cerebellar granule cells contain a unique subset, namely the α1-, α6-, β2-, γ2- and δ-subunits. We studied their GABAergic pharmacology in situ using whole-cell patch-clamp recordings in brain slices and a modified Y-tube application system. The distribution of the EC 50s for GABA in young (P8–P14) and medium aged animals (P15–P28) could be fitted with the sum of two Gaussian distributions with means of 60 and 185 μM and 27 and 214 μM, respectively. In older animals (P29–P48) the observed homogeneous range of sensitivities fitted a single Gaussian distribution (11 μM). In young animals (≤P14) GABA-responses were largely insensitive towards 300 μM of the α6-specific inhibitor furosemide (82% of control response). The sensitivity increased in older animals at the EC 5–20 of GABA (31% of control responses), supporting an increased expression of α6-subunits as molecular basis for the observed developmental changes. Approximately 50% of cells in the age range P15–P48 were potentiated by 1 μM diazepam and by 3 μM methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), suggesting the concurrent presence of α1- and α6-containing receptors, whereas the remaining of cells were neither potentiated by diazepam nor did they show the α6-typical DMCM potentiation, though they were potentiated by loreclezole. These properties indicate unknown pharmacological characteristics of cerebellar receptor-subunit combinations in approximately 50% of granule cells in situ.

Immunocytochemical study of G(i)2alpha and G(o)alpha on the epithelium surface of the rat vomeronasal organ.

To investigate in detail the distribution of G protein subtypes G(i)2alpha and G(o)alpha along the surface of the vomeronasal epithelium, we used double labeling immunocytochemical methods and electron microscopy. We examined the immunoreactivity of these surface structures with antibodies against G(i)2alpha and G(o)alpha. G(i)2alpha- and G(o)alpha-positive cells were observed at the epithelial surface and were evenly distributed. Electron microscopy revealed that strong immunoreactivities to both antibodies were observed on the microvilli and knob-like surface structures of receptor cells. No immunoreactivity was found on the microvilli or surface membranes of supporting cells. This expression pattern is similar to that reported for putative pheromone receptors. These data confirm that there are two distinct classes of vomeronasal receptor cells expressed at the surface of the epithelium. These two classes of receptors correspond to the same G(i)2alpha- and G(o)alpha-positive cells distributed in cell body layers of the epithelium and in the axon terminals in the accessory olfactory bulb.

Distinct classes of human stem cells that differ in proliferative and self-renewal potential.

The composition of the human hematopoietic stem cell compartment is poorly understood due to the absence of experimental tools with which to characterize the developmental program of individual stem cells. We report here that human stem cells differ markedly in their repopulation capacity and self-renewal potential, as determined using nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice transplanted with retrovirally transduced cord blood stem cells, called SCID-repopulating cells (SRCs). Clonal stem cell analysis based on the identification of unique retroviral integration sites within serial bone marrow aspirates showed that repopulation was generally oligoclonal with extensive variability in the lifespan and proliferative capacity of individual SRCs. Most clones contributed to human cell engraftment for several weeks after transplantation and then disappeared but others appeared later and persisted. Further evidence for stem cell heterogeneity was found in the secondary transplantation capacity of SRCs. These data point to the existence of different classes of human stem cells with variable self-renewal potential and short- or long-term repopulating capacity.

The cells of the rabbit meniscus: their arrangement, interrelationship, morphological variations and cytoarchitecture.

Four major morphologically distinct classes of cells were identified within the adult rabbit meniscus using antibodies to cytoskeletal proteins. Two classes of cell were present in the fibrocartilage region of the meniscus. These meniscal cells exhibited long cellular processes that extended from the cell body. A third cell type found in the inner hyaline-like region of the meniscus had a rounded form and lacked projections. A fourth cell type with a fusiform shape and no cytoplasmic projections was found along the superficial regions of the meniscus. Using a monoclonal antibody to connexin 43, numerous gap junctions were observed in the fibrocartilage region, whereas none were seen in cells either from the hyaline-like or the superficial zones of the meniscus. The majority of the cells within the meniscus exhibited other specific features such as primary cilia and 2 centrosomes. The placement of the meniscal cell subtypes as well as their morphology and architecture support the supposition that their specific characteristics underlie the ability of the meniscus to respond to different types of environmental mechanical loads.

The cells of the rabbit meniscus: their arrangement, interrelationship, morphological variations and cytoarchitecture

Four major morphologically distinct classes of cells were identified within the adult rabbit meniscus using antibodies to cytoskeletal proteins. Two classes of cell were present in the fibrocartilage region of the meniscus. These meniscal cells exhibited long cellular processes that extended from the cell body. A third cell type found in the inner hyaline-like region of the meniscus had a rounded form and lacked projections. A fourth cell type with a fusiform shape and no cytoplasmic projections was found along the superficial regions of the meniscus. Using a monoclonal antibody to connexin 43, numerous gap junctions were observed in the fibrocartilage region, whereas none were seen in cells either from the hyaline-like or the superficial zones of the meniscus. The majority of the cells within the meniscus exhibited other specific features such as primary cilia and 2 centrosomes. The placement of the meniscal cell subtypes as well as their morphology and architecture support the supposition that their specific characteristics underlie the ability of the meniscus to respond to different types of environmental mechanical loads.

Structural organization of parallel information processing within the tectofugal visual system of the pigeon.

Visual information processing within the ascending tectofugal pathway to the forebrain undergoes essential rearrangements between the mesencephalic tectum opticum and the diencephalic nucleus rotundus of birds. The outer tectal layers constitute a two-dimensional map of the visual surrounding, whereas nucleus rotundus is characterized by functional domains in which different visual features such as movement, color, or luminance are processed in parallel. Morphologic correlates of this reorganization were investigated by means of focal injections of the neuronal tracer choleratoxin subunit B into different regions of the nuclei rotundus and triangularis of the pigeon. Dependent on the thalamic injection site, variations in the retrograde labeling pattern of ascending tectal efferents were observed. All rotundal projecting neurons were located within the deep tectal layer 13. Five different cell populations were distinguished that could be differentiated according to their dendritic ramifications within different retinorecipient laminae and their axons projecting to different subcomponents of the nucleus rotundus. Because retinorecipient tectal layers differ in their input from distinct classes of retinal ganglion cells, each tectorotundal cell type probably processes different aspects of the visual surrounding. Therefore, the differential input/output connections of the five tectorotundal cell groups might constitute the structural basis for spatially segregated parallel information processing of different stimulus aspects within the tectofugal visual system. Because two of five rotundal projecting cell groups additionally exhibited quantitative shifts along the dorsoventral extension of the tectum, data also indicate visual field-dependent alterations in information processing for particular visual features.

Alterations in electrophysiological activity and dye coupling of striatal spiny and aspiny neurons in dopamine-denervated rat striatum recorded in vivo.

We recently reported that pharmacological manipulations of the dopamine system can produce more than a 4-fold increase in dye coupling between dopaminoceptive neurons in the adult rat striatal complex. During in vivo intracellular recordings, striatal neurons in control rats and in rats that had been treated with 6-hydroxydopamine were injected with either Lucifer yellow or Neurobiotin. Only rats that exhibited severe loss (i.e., larger than approximately 95%) of striatal dopamine terminals displayed a significant increase in the incidence of dye coupling between neurons in adult striatum. Moreover, this increased coupling was present only between neurons of the same morphological cell class, i.e., among clusters of spiny neurons or between aspiny neurons. Combining intracellular labeling of spiny neurons with parvalbumin immunocytochemistry demonstrated that coupling did not occur between anatomically adjacent neurons that comprised immunocytochemically and morphologically distinct cell classes. Therefore, gap junction conductance as reflected by dye coupling appears to undergo upregulation as a consequence of compromises in nigrostriatal and mesolimbic dopamine transmission.

Antigen-Specific Cytolysis by Neutrophils and NK Cells Expressing Chimeric Immune Receptors Bearing ζ or γ Signaling Domains

AbstractTCR- and IgG-binding Fc receptors (FcγR) mediate a variety of critical biologic activities including cytolysis via the structurally related ζ- and γ-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR ζ-chain. Such ζ-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both ζ- and γ-CIRs to activate the cytolytic function of two distinct classes of FcγR-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing ζ- and γ-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both ζ- and γ-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the ζ-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.

Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures

In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2–4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2–4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity—the presumed chick ciliary neurotrophic factor (CNTF)—stimulated the expression of opsin in retinal cultures (EC 50: 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures.

Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression was diffuse throughout the optic vesicles but became restricted to their dorsal part when the vesicles contacted the surface ectoderm. As the optic cup forms, Otx2 was expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2 expression pattern was complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk. Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increased with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate that Otx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes.

Presentation of abundant endogenous class II DR-restricted antigens by DM-negative B cell lines.

Peptides derived from endogenous and exogenous antigens compete for binding and presentation via class II molecules. Studies with mutant B cell lines defective in exogenous antigen presentation suggest that HLA-DM molecules facilitate the interaction of foreign peptides and class II molecules. In contrast, presentation of self antigens is not strictly dependent upon HLA-DM, as demonstrated by the ability of these mutant cells to activate T cells specific for endogenous antigens. Two distinct classes of DM-negative cells, T2 cells generated by in vitro mutagenesis and lines derived from bare lymphocyte syndrome (BLS) patients, were able to present epitopes derived from self proteins. Transfection of DM genes into the mutant cells enhanced the presentation of some, but not all, endogenous antigens, suggesting that formation of select endogenous peptide/class II complexes is not dependent upon DM. The efficiency of endogenous antigen presentation in the absence of DM was also dependent on the mutant antigen-presenting cell studied, as the TxB hybrid T2 presented greater amounts of self peptides compared to cells from BLS patients. Thus, additional genes, aside from DM, may regulate the pathway for endogenous antigen presentation.

システムニューロサイエンス教育講座 ６．舌下神経前位核と眼球運動

This article reviews the nucleus prepositus hypoglossi (NPH) which has been proved to contribute to generation of horizontal eye movements. In general, the NPH receives inputs from oculomotor related nuclei (e.g. the bilateral NPH, the medial vestibular nucleus and ipsilateral burst neuron areas), and projects directly to the bilateral NPH, the vestibular nuclei, the ocular motor nuclei and vestibulo-cerebellar structures. According to these different neural connections, the NPH has been divided into subdivi-sions. Several types of neural activities relating to eye movements are observed in NPH neurons, most of which are increased during ipsiversive horizontal eye movements. The majority of the neurons encode both eye velocity and position signals (burst-tonic neurons). Some of the neurons project to the abducens nucleus. In addition, a class of NPH cells show a burst of activity during a variety of rapid eye movements in cats (burster-driving neurons). These neurons receive disynaptic vestibular input contralaterally and project to burst neuron areas. There are also NPH cells encoding both eye- and head-velocity signals in monkeys (eye-head-velocity neurons). Lesions of the NPH impair gaze holding, implying that the NPH functions as, at least a part of, the neural integrator. However, saccade feedback and putative smooth-pursuit integrators are not influenced after a partial lesion in the NPH. The results suggest that distinct classes of cells spread over the NPH. In summary, the NPH is involved in several aspects of horizontal eye movements and may also function as a part of the neural integrator.

Long-Term Potentiation in Distinct Subtypes of Hippocampal Nonpyramidal Neurons

We have investigated NMDA receptor-dependent long-term potentiation (LTP) in distinct subtypes of nonpyramidal neurons of the CA1 hippocampus using induction protocols that permitted the differentiation between a direct form of LTP and plasticity resulting simply from the "passive propagation" of LTP occurring on CA1 pyramidal neurons. Two types of stratum (st.) oriens/ alveus interneurons received passive propagation of synaptic potentiation via the recurrent collaterals of CA1 pyramidal cells, but neither subtype possessed direct plasticity. In st. radiatum, two distinct classes of cells were observed: st. radiatum interneurons that showed neither direct nor propagated forms of synaptic plasticity, and "giant cells" for which EPSPs were robustly potentiated after a pairing protocol. This potentiation is similar to the LTP described in pyramidal cells, and its induction requires NMDA receptor activation. Thus, a large heterogeneity of synaptic plasticity exists in morphologically distinct neurons and suggests that complex changes in the CA1 network properties will occur after the induction of LTP.

Temporal-frequency selectivity in monkey visual cortex.

We investigated the dynamics of neurons in the striate cortex (V1) and the lateral geniculate nucleus (LGN) to study the transformation in temporal-frequency tuning between the LGN and V1. Furthermore, we compared the temporal-frequency tuning of simple with that of complex cells and direction-selective cells with nondirection-selective cells, in order to determine whether there are significant differences in temporal-frequency tuning among distinct functional classes of cells within V1. In addition, we compared the cells in the primary input layers of V1 (4a, 4c alpha, and 4c beta) with cells in the layers that are predominantly second and higher order (2, 3, 4b, 5, and 6). We measured temporal-frequency responses to drifting sinusoidal gratings. For LGN neurons and simple cells, we used the amplitude and phase of the fundamental response. For complex cells, the elevation of impulse rate (F0) to a drifting grating was the response measure. There is significant low-pass filtering between the LGN and the input layers of V1 accompanied by a small, 3-ms increase in visual delay. There is further low-pass filtering between V1 input layers and the second- and higher-order neurons in V1. This results in an average decrease in high cutoff temporal-frequency between the LGN and V1 output layers of about 20 Hz and an increase in average visual latency of about 12-14 ms. One of the most salient results is the increased diversity of the dynamic properties seen in V1 when compared to the cells of the lateral geniculate, possibly reflecting specialization of function among cells in V1. Simple and complex cells had distributions of temporal-frequency tuning properties that were similar to each other. Direction-selective and nondirection-selective cells had similar preferred and high cutoff temporal frequencies, but direction-selective cells were almost exclusively band-pass while nondirection-selective cells distributed equally between band-pass and low-pass categories. Integration time, a measure of visual delay, was about 10 ms longer for V1 than LGN. In V1 there was a relatively broad distribution of integration times from 40-80 ms for simple cells and 60-100 ms for complex cells while in the LGN the distribution was narrower.

Selective retention of the fluorescent dye DASPEI in a larval gastropod mollusc after paraformaldehyde fixation

In the vertebrates, the vital mitochondrial dye DASPEI (2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide) has been used for the rapid visualization of several distinct classes of epidermal cells in vivo and in vitro: epidermal electroreceptors, mechanoreceptors, and chloride cells in teleosts, and mechanoreceptors in amphibians. I used DASPEI in an attempt to locate a different type of sensory cell, namely, the chemosensory neurons that mediate the initiation of metamorphosis in veliger larvae of the prosobranch gastropod Ilyanassa obsoleta. In vivo, bath-applied DASPEI stains entire larvae in a relatively non-specific fashion. After fixation in phosphate-buffered paraformaldehyde, most of the cells in these animals retained relatively little stain, with some exceptions. Significant DASPEI staining was maintained in approximately nine neurons in the apical ganglion, a temporary cephalic structure that is lost at metamorphosis. Little staining was observed in the rest of the larval central nervous system (CNS), nor could any peripheral sensory neurons be definitively identified. DASPEI was also retained by cells in other larval organs such as the velum and buccal mass, and in two acellular structures, the radula and operculum.

Visually evoked calcium action potentials in cat striate cortex.

Early intracellular studies of cerebral cortical neurons indicated that synaptic input evokes dendritic action potentials that convey information towards the soma. Subsequent work in vitro established that neocortical neurons produce dendritic Ca2+ action potentials. To determine whether natural stimuli elicit Ca2+ spikes, we combined the techniques of whole-cell recording, pharmacology and quantitative receptive field mapping. Our findings show that visual stimulation routinely evoked Ca2+ spikes in distinct functional and anatomical classes of cells in different layers of the cat striate cortex. Hence regenerative Ca2+ potentials appear to play a role in both the initial and later stages of cortical sensory processing.

Pronounced cellular diversity and extrasynaptic location of nicotinic acetylcholine receptor subunit immunoreactivities in the chicken pretectum

The diversity of nicotinic ACh receptor (AChR) expression in the chick lateral spiriform nucleus (SpL) was assessed using subunit-specific monoclonal antibodies (mAbs) and laser scanning confocal microscopy. The late embryonic SpL was immunoreactive for mAbs against the alpha 2, alpha 5, alpha 7, alpha 8, and beta 2 AChR subunits. Distinct neuronal cell classes were determined using pair-wise staining of mAbs. Approximately 90% of the neurons in the SpL contained both alpha 5-like immunoreactivity (LI) and beta 2-LI, with no neurons having only one of these subunit-LIs. Approximately 70% of the neurons contained alpha 2-LI. All alpha 2-LI neurons contained alpha 5/beta 2-LI; thus, neurons having alpha 2-LI are a subset of those having alpha 5- and beta 2-LI. Fewer neurons, approximately 20%, contained alpha 7-LI. A subset of alpha 7-positive neurons were immunoreactive for other subunits; for example, some alpha 7-positive neurons also contained alpha 2-LI. Fewer than 15% of the neurons contained alpha 8-LI. Some of the alpha 8-LI-containing neurons contained alpha 7-LI. The 14 week post-hatch SpL resembles the late embryonic nucleus in the percentage of neurons immunoreactive for alpha 2, alpha 5, alpha 7, alpha 8, and beta 2 AChR subunits, and in the presence of multiple classes based on AChR subunit immunoreactivity. In addition, alpha 4-LI was found in about 20% of the 14 week SpL neurons. Double-label immunofluorescence experiments with mAbs to AChRs and to synaptic vesicle antigens showed that most clusters of alpha 5-LI and beta 2-LI are extrasynaptic. The pronounced diversity of AChR subunit expression and the extrasynaptic location of AChR-LI suggest that AChR-like molecules in the SpL do not function solely to respond to transmitter focally released from presynaptic terminals.