Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Cell-Type-Specific Whole-Brain Direct Inputs to the Anterior and Posterior Piriform Cortex.

The piriform cortex (PC) is a key brain area involved in both processing and coding of olfactory information. It is implicated in various brain disorders, such as epilepsy, Alzheimer's disease, and autism. The PC consists of the anterior (APC) and posterior (PPC) parts, which are different anatomically and functionally. However, the direct input networks to specific neuronal populations within the APC and PPC remain poorly understood. Here, we mapped the whole-brain direct inputs to the two major neuronal populations, the excitatory glutamatergic principal neurons and inhibitory γ-aminobutyric acid (GABA)-ergic interneurons within the APC and PPC using the rabies virus (RV)-mediated retrograde trans-synaptic tracing system. We found that for both types of neurons, APC and PPC share some similarities in input networks, with dominant inputs originating from the olfactory region (OLF), followed by the cortical subplate (CTXsp), isocortex, cerebral nuclei (CNU), hippocampal formation (HPF) and interbrain (IB), whereas the midbrain (MB) and hindbrain (HB) were rarely labeled. However, APC and PPC also show distinct features in their input distribution patterns. For both types of neurons, the input proportion from the OLF to the APC was higher than that to the PPC; while the PPC received higher proportions of inputs from the HPF and CNU than the APC did. Overall, our results revealed the direct input networks of both excitatory and inhibitory neuronal populations of different PC subareas, providing a structural basis to analyze the diverse PC functions.

Diverse glutamatergic inputs target spines expressing M1 muscarinic receptors in the basolateral amygdala: An ultrastructural analysis.

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.

Architectonic features and relative locations of primary sensory and related areas of neocortex in mouse lemurs.

Mouse lemurs are the smallest of the living primates, and are members of the understudied radiation of strepsirrhine lemurs of Madagascar. They are thought to closely resemble the ancestral primates that gave rise to present day primates. Here we have used multiple histological and immunochemical methods to identify and characterize sensory areas of neocortex in four brains of adult lemurs obtained from a licensed breeding colony. We describe the laminar features for the primary visual area (V1), the secondary visual area (V2), the middle temporal visual area (MT) and area prostriata, somatosensory areas S1(3b), 3a, and area 1, the primary motor cortex (M1), and the primary auditory cortex (A1). V1 has "blobs" with "nonblob" surrounds, providing further evidence that this type of modular organization might have evolved early in the primate lineage to be retained in all extant primates. The laminar organization of V1 further supports the view that sublayers of layer 3 of primates have been commonly misidentified as sublayers of layer 4. S1 (area 3b) is proportionately wider than the elongated area observed in anthropoid primates, and has disruptions that may distinguish representations of the hand, face, teeth, and tongue. Primary auditory cortex is located in the upper temporal cortex and may include a rostral area, R, in addition to A1. The resulting architectonic maps of cortical areas in mouse lemurs can usefully guide future studies of cortical connectivity and function.

Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli.

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

Protein Markers of Neurotransmitter Synthesis and Release in Postmortem Schizophrenia Substantia Nigra.

The substantia nigra (SN) provides the largest dopaminergic input to the brain, projects to the striatum (the primary locus of action for antipsychotic medication), and receives GABAergic and glutamatergic inputs. This study used western blot analysis to compare protein levels of tyrosine hydroxylase (TH), glutamate decarboxylase (GAD67), and vesicular glutamate transporters (vGLUT1 and vGLUT2) in postmortem human SN in schizophrenia subjects (n=13) and matched controls (n=12). As a preliminary analysis, the schizophrenia group was subdivided by (1) treatment status: off medication (n=4) or on medication (n=9); or (2) treatment response: treatment resistant (n=5) or treatment responsive (n=4). The combined schizophrenia group had higher TH and GAD67 protein levels than controls (an increase of 69.6%, P=0.01 and 19.5%, P=0.004, respectively). When subdivided by medication status, these increases were found in the on-medication subjects (TH 88.3%, P=0.008; GAD67 40.6%, P=0.003). In contrast, unmedicated schizophrenia subjects had higher vGLUT2 levels than controls (an increase of 28.7%, P=0.041), but vGLUT2 levels were similar between medicated schizophrenia subjects and controls. Treatment-resistant subjects had significantly higher TH and GAD67 levels than controls (an increase of 121.0%, P=0.0003 and 58.7%, P=0.004, respectively). These data suggest increases in dopamine and GABA transmission in the SN in schizophrenia, with a potential relation to treatment and response.

Ultrastructure of geniculocortical synaptic connections in the tree shrew striate cortex.

To determine whether thalamocortical synaptic circuits differ across cortical areas, we examined the ultrastructure of geniculocortical terminals in the tree shrew striate cortex to compare directly the characteristics of these terminals with those of pulvinocortical terminals (examined previously in the temporal cortex of the same species; Chomsung et al. [] Cereb Cortex 20:997-1011). Tree shrews are considered to represent a prototype of early prosimian primates but are unique in that sublaminae of striate cortex layer IV respond preferentially to light onset (IVa) or offset (IVb). We examined geniculocortical inputs to these two sublayers labeled by tracer or virus injections or an antibody against the type 2 vesicular glutamate antibody (vGLUT2). We found that layer IV geniculocortical terminals, as well as their postsynaptic targets, were significantly larger than pulvinocortical terminals and their postsynaptic targets. In addition, we found that 9-10% of geniculocortical terminals in each sublamina contacted GABAergic interneurons, whereas pulvinocortical terminals were not found to contact any interneurons. Moreover, we found that the majority of geniculocortical terminals in both IVa and IVb contained dendritic protrusions, whereas pulvinocortical terminals do not contain these structures. Finally, we found that synaptopodin, a protein uniquely associated with the spine apparatus, and telencephalin (TLCN, or intercellular adhesion molecule type 5), a protein associated with maturation of dendritic spines, are largely excluded from geniculocortical recipient layers of the striate cortex. Together our results suggest major differences in the synaptic organization of thalamocortical pathways in striate and extrastriate areas.

Tyramide Signal Amplification for Immunofluorescent Enhancement.

Enzyme-linked signal amplification is a key technique used to enhance the immunohistochemical detection of protein, mRNA, and other molecular species. Tyramide signal amplification (TSA) is based on a catalytic reporter deposit in close vicinity to the epitope of interest. The advantages of this technique are its simplicity, enhanced sensitivity, high specificity, and compatibility with modern multi-label fluorescent microscopy. Here, we describe the use of a TSA kit to increase the signal of enhanced green fluorescent protein (eGFP) expressed under the control of Slc17a6 regulatory elements in the brain of a transgenic mouse. The labeling procedure consists of 6 basic steps: (1) tissue preparation, (2) blocking of nonspecific epitopes, (3) binding with primary antibody, (4) binding with horseradish peroxidase-conjugated secondary antibody, (5) reacting with fluorescent tyramide substrate, and (6) imaging of the signal. The procedures described herein detail these steps and provide additional guidance and background to assist novice users.

The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping.

Amylin reduces meal size by activating noradrenergic neurons in the area postrema (AP). Neurons in the AP also mediate the eating-inhibitory effects of salmon calcitonin (sCT), a potent amylin agonist, but the phenotypes of the neurons mediating its effect are unknown. Here we investigated whether sCT activates similar neuronal populations to amylin, and if its anorectic properties also depend on AP function. Male rats underwent AP lesion (APX) or sham surgery. Meal patterns were analysed under ad libitum and post-deprivation conditions. The importance of the AP in mediating the anorectic action of sCT was examined in feeding experiments of dose-response effects of sCT in APX vs. sham rats. The effect of sCT to induce Fos expression was compared between surgery groups, and relative to amylin. The phenotype of Fos-expressing neurons in the brainstem was examined by testing for the co-expression of dopamine beta hydroxylase (DBH) or tryptophan hydroxylase (TPH). By measuring the apposition of vesicular glutamate transporter-2 (VGLUT2)-positive boutons, potential glutamatergic input to amylin- and sCT-activated AP neurons was compared. Similar to amylin, an intact AP was necessary for sCT to reduce eating. Further, co-expression between Fos activation and DBH after amylin or sCT did not differ markedly, while co-localization of Fos and TPH was minor. Approximately 95% of neurons expressing Fos and DBH after amylin or sCT treatment were closely apposed to VGLUT2-positive boutons. Our study suggests that the hindbrain pathways engaged by amylin and sCT share many similarities, including the mediation by AP neurons.

Loss of corticostriatal and thalamostriatal synaptic terminals precedes striatal projection neuron pathology in heterozygous Q140 Huntington's disease mice.

Motor slowing, forebrain white matter loss, and striatal shrinkage have been reported in premanifest Huntington's disease (HD) prior to overt striatal neuron loss. We carried out detailed LM and EM studies in a genetically precise HD mimic, heterozygous Q140 HD knock-in mice, to examine the possibility that loss of corticostriatal and thalamostriatal terminals prior to striatal neuron loss underlies these premanifest HD abnormalities. In our studies, we used VGLUT1 and VGLUT2 immunolabeling to detect corticostriatal and thalamostriatal (respectively) terminals in dorsolateral (motor) striatum over the first year of life, prior to striatal projection neuron pathology. VGLUT1+ axospinous corticostriatal terminals represented about 55% of all excitatory terminals in striatum, and VGLUT2+ axospinous thalamostriatal terminals represented about 35%, with VGLUT1+ and VGLUT2+ axodendritic terminals accounting for the remainder. In Q140 mice, a significant 40% shortfall in VGLUT2+ axodendritic thalamostriatal terminals and a 20% shortfall in axospinous thalamostriatal terminals were already observed at 1 month of age, but VGLUT1+ terminals were normal in abundance. The 20% deficiency in VGLUT2+ thalamostriatal axospinous terminals persisted at 4 and 12 months in Q140 mice, and an additional 30% loss of VGLUT1+ corticostriatal terminals was observed at 12 months. The early and persistent deficiency in thalamostriatal axospinous terminals in Q140 mice may reflect a development defect, and the impoverishment of this excitatory drive to striatum may help explain early motor defects in Q140 mice and in premanifest HD. The loss of corticostriatal terminals at 1 year in Q140 mice is consistent with prior evidence from other mouse models of corticostriatal disconnection early during progression, and can explain both the measurable bradykinesia and striatal white matter loss in late premanifest HD.

Muscarinic acetylcholine receptors in the mouse esophagus: focus on intraganglionic laminar endings (IGLEs).

BACKGROUND: IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. METHODS: This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. KEY RESULTS: Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. CONCLUSIONS & INFERENCES: Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.

Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice - effects of peripheral axotomy or hindpaw inflammation.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

Projections of the superior colliculus to the pulvinar in prosimian galagos (Otolemur garnettii) and VGLUT2 staining of the visual pulvinar.

An understanding of the organization of the pulvinar complex in prosimian primates has been somewhat elusive due to the lack of clear architectonic divisions. In the current study we reveal features of the organization of the pulvinar complex in galagos by examining superior colliculus (SC) projections to this structure and comparing them with staining patterns of the vesicular glutamate transporter, VGLUT2. Cholera toxin subunit ß (CTB), Fluoro-ruby (FR), and wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were placed in topographically different locations within the SC. Our results showed multiple topographically organized patterns of projections from the SC to several divisions of the pulvinar complex. At least two topographically distributed projections were found within the lateral region of the pulvinar complex, and two less obvious topographical projection patterns were found within the caudomedial region, in zones that stain darkly for VGLUT2. The results, considered in relation to recent observations in tree shrews and squirrels, suggest that parts of the organizational scheme of the pulvinar complex in primates are present in rodents and other mammals.

Evidence for ape and human specializations in geniculostriate projections from VGLUT2 immunohistochemistry.

Vesicular glutamate transporters (VGLUTs) reuptake glutamate into synaptic vesicles at excitatory synapses. VGLUT2 is localized in the cortical terminals of neuronal somas located in the main sensory nuclei of the thalamus. Thus, immunolabeling of cortex with antibodies to VGLUT2 can reveal geniculostriate terminal distributions in species in which connectivity cannot be studied with tract-tracing techniques, permitting broader comparative studies of cortical specializations. Here, we used VGLUT2 immunohistochemistry to compare the organization of geniculostriate afferents in primary visual cortex in hominid primates (humans, chimpanzees, and an orangutan), Old World monkeys (rhesus macaques and vervets), and New World monkeys (squirrel monkeys). The New and Old World monkeys had a broad, dense band of terminal-like labeling in cortical layer 4C, a narrow band of labeling in layer 4A, and additional labeling in layers 2/3 and 6, consistent with results from conventional tract-tracing studies in these species. By contrast, although the hominid primates had a prominent layer 4C band, labeling of layer 4A was sparse or absent. Labeling was also present in layers 2/3 and 6, although labeling of layer 6 was weaker in hominids and possibly more individually variable than in Old and New World monkeys. These findings are consistent with previous observations from cytochrome oxidase histochemistry and a very small number of connectivity studies, suggesting that the projections from the parvocellular layers of the lateral geniculate nucleus to layer 4A were strongly reduced or eliminated in humans and apes following their evolutionary divergence from the other anthropoid primates.

Inner hair cells of mice express the glutamine transporter SAT1.

Glutamate has been implicated in signal transmission between inner hair cells and afferent fibers of the organ of Corti. The inner hair cells are enriched in glutamate and the postsynaptic membranes express AMPA glutamate receptors. However, it is not known whether inner hair cells contain a mechanism for glutamate replenishment. Such a mechanism must be in place to sustain glutamate neurotransmission. Here we provide RT-PCR and immunofluorescence data indicating that system A transporter 1 (SLC38A1), which is associated with neuronal glutamine transport and synthesis of the neurotransmitters GABA and glutamate in CNS, is expressed in inner hair cells. It was previously shown that inner hair cells contain glutaminase that converts glutamine to glutamate. Thus, our finding that inner hair cells express a glutamine transporter and the key glutamine metabolizing enzyme glutaminase, provides a mechanism for glutamate replenishment and bolsters the idea that glutamate serves as a transmitter in the peripheral synapse of the auditory system.

Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes.

The brainstem premotor neurons of the facial nucleus (VII) and hypoglossal (XII) nucleus can integrate orofacial nociceptive input from the caudal spinal trigeminal nucleus (Vc) and coordinate orofacial nociceptive reflex (ONR) responses. However, the synaptoarchitectures of the ONR pathways are still unknown. In the current study, we examined the distribution of GABAergic premotor neurons in the brainstem local ONR pathways, their connections with the Vc projections joining the brainstem ONR pathways and the neurochemical properties of these connections. Retrograde tracer fluoro-gold (FG) was injected into the VII or XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the Vc. Immunofluorescence histochemical labeling for inhibitory/excitatory neurotransmitters combined with BDA/FG tracing showed that GABAergic premotor neurons were mainly distributed bilaterally in the ponto-medullary reticular formation with an ipsilateral dominance. Some GABAergic premotor neurons made close appositions to the BDA-labeled fibers coming from the Vc, and these appostions were mainly distributed in the parvicellular reticular formation (PCRt), dorsal medullary reticular formation (MdD), and supratrigeminal nucleus (Vsup). We further examined the synaptic relationships between the Vc projecting fibers and premotor neurons in the VII or XII under the confocal laser-scanning microscope and electron microscope, and found that the BDA-labeled axonal terminals that made asymmetric synapses on premotor neurons showed vesicular glutamate transporter 2 (VGluT2) like immunoreactivity. These results indicate that the GABAergic premotor neurons receive excitatory neurotransmission from the Vc and may contribute to modulating the generation of the tonic ONR.

Vesicular glutamate transporters in axons that innervate the human dental pulp.

INTRODUCTION: Vesicular glutamate transporters (VGLUTs) are involved in the transport of transmitter glutamate into synaptic vesicles and are used as markers for glutamatergic neurons. METHODS: To assess which types of VGLUTs are involved in the glutamate signaling in pulpal axons and to investigate their distribution, we performed light microscopic immunohistochemistry by using antibodies against VGLUT1, VGLUT2, calcitonin gene-related peptide, and Western blot analysis in human dental pulp. RESULTS: VGLUT1 was expressed in a large number of pulpal axons, especially in the peripheral pulp where the axons branch extensively. The VGLUT1 immunopositive axons showed bead-like appearance, and the majority of these also expressed calcitonin gene-related peptide. VGLUT2 was expressed in few axons throughout the pulp. CONCLUSIONS: Our findings suggest that VGLUT1 is involved mainly in the glutamate-mediated signaling of pain, primarily at the level of the peripheral pulp.

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.

A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Rapid structural remodeling of thalamocortical synapses parallels experience-dependent functional plasticity in mouse primary visual cortex.

Monocular lid closure (MC) causes a profound shift in the ocular dominance (OD) of neurons in primary visual cortex (V1). Anatomical studies in both cat and mouse V1 suggest that large-scale structural rearrangements of eye-specific thalamocortical (TC) axons in response to MC occur much more slowly than the shift in OD. Consequently, there has been considerable debate as to whether the plasticity of TC synapses, which transmit competing visual information from each eye to V1, contributes to the early functional consequences of MC or is simply a feature of long-term deprivation. Here, we used quantitative immuno-electron microscopy to examine the possibility that alterations of TC synapses occur rapidly enough to impact OD after brief MC. The effect of short-term deprivation on TC synaptic structure was examined in male C57BL/6 mice that underwent 3 and 7 d of MC or monocular retinal inactivation (MI) with tetrodotoxin. The data show that 3 d of MC is sufficient to induce substantial remodeling of TC synapses. In contrast, 3 d of MI, which alters TC activity but does not shift OD, does not significantly affect the structure of TC synapses. Our results support the hypothesis that the rapid plasticity of TC synapses is a key step in the sequence of events that shift OD in visual cortex.