VGluT1 Deficiency Impairs Visual Attention and Reduces the Dynamic Range of Short-Term Plasticity at Corticothalamic Synapses.

The most common excitatory neurotransmitter in the central nervous system, glutamate, is loaded into synaptic vesicles by vesicular glutamate transporters (VGluTs). The primary isoforms, VGluT1 and 2, are expressed in complementary patterns throughout the brain and correlate with short-term synaptic plasticity. VGluT1 deficiency is observed in certain neurological disorders, and hemizygous (VGluT1+/-) mice display increased anxiety and depression, altered sensorimotor gating, and impairments in learning and memory. The synaptic mechanisms underlying these behavioral deficits are unknown. Here, we show that VGluT1+/- mice had decreased visual processing speeds during a sustained visual-spatial attention task. Furthermore, in vitro recordings of corticothalamic (CT) synapses revealed dramatic reductions in short-term facilitation, increased initial release probability, and earlier synaptic depression in VGluT1+/- mice. Our electron microscopy results show that VGluT1 concentration is reduced at CT synapses of hemizygous mice, but other features (such as vesicle number and active zone size) are unchanged. We conclude that VGluT1-haploinsuficiency decreases the dynamic range of gain modulation provided by CT feedback to the thalamus, and this deficiency contributes to the observed attentional processing deficit. We further hypothesize that VGluT1 concentration regulates release probability by applying a "brake" to an unidentified presynaptic protein that typically acts as a positive regulator of release.

Synaptic Organization of VGLUT3 Expressing Low-Threshold Mechanosensitive C Fiber Terminals in the Rodent Spinal Cord.

Low-threshold mechanosensitive C fibers (C-LTMRs) that express the vesicular glutamate transporter VGLUT3 are thought to signal affective touch, and may also play a role in mechanical allodynia. However, the nature of the central termination of C-LTMRs in the dorsal horn remains largely unexplored. Here, we used light and electron microscopy in combination with VGLUT3 immunolabeling as a marker of C-LTMR terminations to investigate this issue. VGLUT3+ C-LTMRs formed central terminals of Type II glomeruli in the inner part of lamina II of the dorsal horn, often establishing multiple asymmetric synapses with postsynaptic dendrites but also participating in synaptic configurations with presynaptic axons and dendrites. Unexpectedly, essentially all VGLUT3+ C-LTMR terminals showed substantial VGLUT1 expression in the rat, whereas such terminals in mice lacked VGLUT1. Most VGLUT3+ C-LTMR terminals exhibited weak-to-moderate VGLUT2 expression. Further, C-LTMR terminals formed numerous synapses with excitatory protein kinase Cγ (PKCγ) interneurons and inhibitory parvalbumin neurons, whereas synapses with calretinin neurons were scarce. C-LTMR terminals rarely if ever established synapses with neurokinin 1 receptor (NK1R)-possessing dendrites traversing lamina II. Thus, VGLUT3+ C-LTMR terminals appear to largely correspond to neurofilament-lacking central terminals of Type II glomeruli in inner lamina II and can thus be identified at the ultrastructural level by morphological criteria. The participation of C-LTMR terminals in Type II glomeruli involving diverse populations of interneuron indicates highly complex modes of integration of C-LTMR mediated signaling in the dorsal horn. Furthermore, differences in VGLUT1 expression indicate distinct species differences in synaptic physiology of C-LTMR terminals.

Towards a Terminologia Neuroanatomica.

This article deals with a recent revision of the terminology of the Sections Central Nervous System (CNS; Systema nervosum centrale) and Peripheral Nervous System (PNS; Systema nervosum periphericum) of the Terminologia Anatomica (TA, 1998) and the Terminologia Histologica (TH, 2008). These sections were extensively updated by the Federative International Programme for Anatomical Terminology (FIPAT) Working Group Neuroanatomy of the International Federation of Associations of Anatomists (IFAA). After extensive discussions by FIPAT, and consultation with the IFAA Member Societies, these parts were merged to form a Terminologia Neuroanatomica (TNA). After validation at the IFAA Executive Meeting, September 22, 2016, the TNA has been placed on the open part of the FIPAT website (http://FIPAT.library.dal.ca) as the official FIPAT Terminology. This article outlines the major differences between the TNA and the TA. Clin. Anat. 30:145-155, 2017. © 2016 Wiley Periodicals, Inc.

Locomotor training maintains normal inhibitory influence on both alpha- and gamma-motoneurons after neonatal spinal cord transection.

Spinal cord injuries lead to impairments, which are accompanied by extensive reorganization of neuronal circuits caudal to the injury. Locomotor training can aid in the functional recovery after injury, but the neuronal mechanisms associated with such plasticity are only sparsely known. We investigated ultrastructurally the synaptic inputs to tibialis anterior motoneurons (MNs) retrogradely labeled in adult rats that had received a complete midthoracic spinal cord transection at postnatal day 5. A subset of the injured rats received locomotor training. Both γ- and α-MNs were studied. The total number of boutons apposing γ-MNs, but not α-MNs, was reduced after neonatal spinal cord transection. The proportion of inhibitory to excitatory boutons, however, was increased significantly in both α-MNs and γ-MNs in spinally transected rats, but with locomotor training returned to levels observed in intact rats. The specific densities and compositions of synaptic boutons were, however, different between all three groups. Surprisingly, we observed the atypical presence of both C- and M-type boutons apposing the somata of γ-MNs in the spinal rats, regardless of training status. We conclude that a neonatal spinal cord transection induces significant reorganization of synaptic inputs to spinal motoneurons caudal to the site of injury with a net increase in inhibitory influence, which is associated with poor stepping. Spinal cord injury followed by successful locomotor training, however, results in improved bipedal stepping and further synaptic changes with the proportion of inhibitory and excitatory inputs to the motoneurons being similar to that observed in intact rats.

Synaptic plasticity and pain: role of ionotropic glutamate receptors.

Pain hypersensitivity that develops after tissue or nerve injury is dependent both on peripheral processes in the affected tissue and on enhanced neuronal responses in the central nervous system, including the dorsal horn of the spinal cord. It has become increasingly clear that strengthening of glutamatergic sensory synapses, such as those established in the dorsal horn by nociceptive thin-caliber primary afferent fibers, is a major contributor to sensitization of neuronal responses that leads to pain hypersensitivity. Here, the authors review recent findings on the roles of ionotropic glutamate receptors in synaptic plasticity in the dorsal horn in relation to acute and persistent pain.

Translocation of GluR1-containing AMPA receptors to a spinal nociceptive synapse during acute noxious stimulation.

Potentiation of spinal nociceptive transmission by synaptic delivery of AMPA receptors, via an NMDA receptor- and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent pathway, has been proposed to underlie certain forms of hyperalgesia, the enhanced pain sensitivity that may accompany inflammation or tissue injury. However, the specific synaptic populations that may be subject to such plasticity have not been identified. Using neuronal tracing and postembedding immunogold labeling, we show that a model of acute inflammatory hyperalgesia is associated with an elevated density of GluR1-containing AMPA receptors, as well as an increased synaptic ratio of GluR1 to GluR2/3 subunits, at synapses established by C-fibers that lack the neuropeptide substance P. A more subtle increase in GluR1 immunolabeling was noted at synapses formed by substance P-containing nociceptors. No changes in either GluR1 or GluR2/3 contents were observed at synapses formed by low-threshold mechanosensitive primary afferent fibers. These results contrast with our previous observations in the same pain model of increased and decreased levels of activated CaMKII at synapses formed by peptidergic and nonpeptidergic nociceptive fibers, respectively, suggesting that the observed redistribution of AMPA receptor subunits does not depend on postsynaptic CaMKII activity. The present ultrastructural evidence of topographically specific, activity-dependent insertion of GluR1-containing AMPA receptors at a central synapse suggests that potentiation of nonpeptidergic C-fiber synapses by this mechanism contributes to inflammatory pain.

Central projections of the sensory innervation of the rat middle meningeal artery.

Headaches, especially migraine, involve not only pain but also aspects such as vasodilation of cranial vessels and sensitization of nerve endings, processes dependent on and connected to the central nervous system. To understand pathogenic mechanisms of headache, it is important to elucidate the central projections of sensory nerves that innervate cranial vessels, of which the middle meningeal artery (MMA) is the largest artery supplying the dura mater. In this study, cholera toxin subunit b (CTb) or wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) was applied on the adventitia of MMA. After perfusion fixation, the brainstem, the C1-C4 spinal segments and the trigeminal and C2 dorsal root ganglia were removed and sections from these tissues were processed to visualize transported tracers. Labeled cell bodies were seen ipsilaterally in the trigeminal and C2 dorsal root ganglia. Labeled nerve terminations were found ipsilaterally in the lateral part of the spinal dorsal horn of segments C1-C3 and in the caudal and interpolar parts of the spinal trigeminal nucleus. WGA-HRP labeled terminations were mainly located in laminae I and II, whereas CTb labeled terminations located in laminae III-V. These results indicate that sensory information from the MMA is transmitted through both trigeminal and cervical spinal nerve branches to a region in the central nervous system extending rostrally from the C3 dorsal horn to the interpolar part of the spinal trigeminal nucleus. Our data further substantiates that the sensory innervation of the MMA, in addition to putative nociceptive afferents, include a population of large caliber afferents with an as yet unclear but presumably non-nociceptive role.

Expression of calcium channel CaV1.3 in cat spinal cord: light and electron microscopic immunohistochemical study.

In spinal neurons, plateau potentials serve to amplify neuronal input signals. To a large extent, the underlying persistent inward current is mediated by a subtype of the L-type calcium channel (Ca(V)1.3). In the present investigation, we have studied its distribution and cellular localization in the cat spinal cord by light and electron microscopic immunohistochemistry. The results show that Ca(V)1.3-like immunoreactivity is widely distributed in all segments of the spinal cord but that the distribution in the different laminae of the spinal gray matter varies, with the highest density of labeled neurons in lamina IX and the lowest in lamina II. The labeling intensity was highest in neuronal somata, but a certain length of the proximal dendrite was also labeled. Some neuronal groups exhibited a particularly dense labeling; these include the lateral motoneuronal group in the cervical and the lumbar enlargements and the phrenic nucleus in cervical, Clarke's nucleus in lower thoracic and upper lumbar, and Onuf's nucleus in upper sacral segments. At the ultrastructural level, Ca(V)1.3-immunoreactive products were found in neuronal somata and dendrites of different sizes. In the soma, they were predominantly associated with the rough endoplasmic reticulum but some also with the plasma membrane. In dendrites, they were associated with both intracellular organelles, including microtubules and microchondria, and the plasma membrane. These results indicate that significant proportions of the neurons in cat spinal cord, including projection neurons, interneurons, and motoneurons, are endowed with ion channels that subserve persistent inward currents and act to amplify synaptic input signals.

Time course of cerebellar morphological development in postnatal ferrets: ontogenetic and comparative perspectives.

We provide the first systematic description of the morphological ontogenesis of the ferret cerebellum and compare its relative time-course to that of the rat cerebellum. Overall cerebellar size, foliation, and thickness of cortical layers were quantified and Purkinje cell morphology was characterized at 24 timepoints in ferrets from postnatal day (P)1 to P63. The ferret cerebellum was substantially larger than that of the rat and had a much longer developmental period. In ferrets, Purkinje cells were dispersed into a monolayer by P9, the formation of folia declined abruptly around P20, and the external granular layer peaked in thickness around P22 and disappeared by P56. Timepoints of corresponding relative developmental maturity of the quantified architectural features of rat and ferret cerebella were determined and their relationship was analyzed by linear regression. The time-conversion equation derived, describing the relationship between cerebellar morphogenesis in the two species, had a determination coefficient (r2) of 0.95. Conspicuously, the equation predicted with high accuracy the timing of structural changes in individual Purkinje cells in the ferret cerebellum. The conversion equation should be useful for precise quantitative translation of data between studies of ferret and rat cerebellum and for comparisons between development of motor and sensory structures and functions in ferrets. The degree of similarity in the time-courses of cerebellar development in two distantly related mammals makes explicit in quantitative terms how remarkably conserved the cerebellum is in phylogenesis. Therefore, the methodology should be applicable to precise quantitative conversions of cerebellar developmental time-courses also between other species.

Demonstration of mitochondrial oestrogen receptor beta and oestrogen-induced attenuation of cytochrome c oxidase subunit I expression in human periodontal ligament cells.

OBJECTIVE: Periodontal ligament (PDL) cells express oestrogen receptor beta (ERbeta) protein, but cellular functions regulated by ERbeta in these cells have not been identified. In this study we determine if ERbeta is localised to mitochondria and if oestrogen regulates mitochondrial function in human PDL cells obtained from teeth extracted for orthodontic reasons. DESIGN: Subcellular distribution of ERbeta was determined by confocal microscopy of cells co-stained with ERbeta antibody and the mitochondrion-selective probe MitoTracker and by immunogold electron microscopy. Expression of the mitochondrial enzyme cytochrome c oxidase subunit I, involved in oxidative phosphorylation, was determined by Western blotting in cells treated with or without physiological concentrations of the endogenous oestrogen 17beta-oestradiol. RESULTS: ERbeta immunoreactivity was observed both in the nuclei and the cytoplasm. MitoTracker-labelling was observed in the cytoplasm, especially in the perinuclear region, but not in the nuclei. Co-localisation of ERbeta and MitoTracker was observed in cells derived from both male and female subjects. Mitochondrial localisation of ERbeta was confirmed by immunogold electron microscopy. Cells treated with or without 17beta-oestradiol (100 nM) displayed an identical pattern of staining for mitochondria. Treatment with 100 nM 17beta-oestradiol attenuated cytochrome c oxidase subunit I expression by about 30%, while combined treatment with 17beta-oestradiol and the ER blocker ICI 182780 (10 microM) had no effect. CONCLUSION: This study demonstrates mitochondrial localisation of ERbeta and oestrogen-induced decrease in the expression of cytochrome c oxidase subunit I in human PDL cells, suggesting that oestrogen probably via ERbeta influences mitochondrial function and PDL cell energy metabolism.

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1.

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca(2+) sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were approximately 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased alpha(1)-adrenergic contraction. Following inhibition of PKC, alpha(1)-adrenergic contraction was normalized. PDBu-induced Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca(2+) sensitivity.

Ultrastructural synaptic features differ between alpha- and gamma-motoneurons innervating the tibialis anterior muscle in the rat.

We investigated the synaptology of retrogradely labeled spinal motoneurons after injection of horseradish peroxidase into the tibialis anterior (TA) muscle of adult rat. In total, 32 TA motoneurons were investigated in the electron microscope and demonstrated a bimodal size distribution with cell diameter peaks at 40 microm and 20 microm, likely representing alpha- and gamma-motoneurons, respectively. Both alpha- and gamma-motoneurons were apposed by S- and F-type synaptic boutons, whereas only alpha-motoneurons demonstrated inputs by the large M- and C-type boutons. The proportion of cell body membrane covered by synaptic inputs was surprisingly indistinguishable between alpha-motoneurons (72.2%) and gamma-motoneurons (63.5%). The ratio between the number of F- and S-type boutons in apposition with the motoneuron cell body (F/S ratio) and the ratio between the soma membrane coverage provided by F- and S-type boutons were both significantly higher in alpha- than in gamma-motoneurons. When comparing our data with previous findings in other species, we conclude that rat TA alpha-motoneurons are similar to cat and primate alpha-motoneurons with regard to synaptic terminal morphology, frequency, and distribution. However, rat gamma-motoneurons show a markedly higher total synaptic coverage and frequency than cat gamma-motoneurons, although both species exhibit appositions made by the same synaptic types and similar ratios between inhibitory and excitatory inputs.

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract.

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.

Pathway-specific bidirectional regulation of Ca2+/calmodulin-dependent protein kinase II at spinal nociceptive synapses after acute noxious stimulation.

An intensely painful stimulus may lead to hyperalgesia, the enhanced sensation of subsequent painful stimuli. This is commonly believed to involve facilitated transmission of sensory signals in the spinal cord, possibly by a long-term potentiation-like mechanism. However, plasticity of identified synapses in intact hyperalgesic animals has not been reported. Here, we show, using neuronal tracing and postembedding immunogold labeling, that after acute noxious stimulation (hindpaw capsaicin injections), immunolabeling of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and of CaMKII phosphorylated at Thr(286/287) (pCaMKII) are upregulated postsynaptically at synapses established by peptidergic primary afferent fibers in the superficial dorsal horn of intact rats. In contrast, postsynaptic pCaMKII immunoreactivity was instead downregulated at synapses of nonpeptidergic primary afferent C-fibers; this loss of pCaMKII immunolabel occurred selectively at distances greater than approximately 20 nm from the postsynaptic membrane and was accompanied by a smaller reduction in total CaMKII contents of these synapses. Both pCaMKII and CaMKII immunogold labeling were unaffected at synapses formed by presumed low-threshold mechanosensitive afferent fibers. Thus, distinct molecular modifications, likely indicative of plasticity of synaptic strength, are induced at different populations of presumed nociceptive primary afferent synapse by intense noxious stimulation, suggesting a complex modulation of parallel nociceptive pathways in inflammatory hyperalgesia. Furthermore, the activity-induced loss of certain postsynaptic pools of autophosphorylated CaMKII at previously unmanipulated synapses supports a role for the kinase in basal postsynaptic function.

Systemic administration of cholera toxin B subunit conjugated to horseradish peroxidase in the adult rat labels preganglionic autonomic neurons, motoneurons, and select primary afferents for light and electron microscopic studies.

Retrograde and transganglionic labeling techniques are commonly used to visualize subsets of neurons and sensory afferent projections in the nervous system. These methods commonly require anesthesia and surgical methods. However, some tracers can also be administered systemically in awake animals, thus reducing risks associated with anesthesia and surgery and allowing for labeling of neuronal populations that are difficult to label with local tracer injections. Here, we demonstrate in the adult rat that intraperitoneal administration of cholera toxin subunit B conjugated to horseradish peroxidase labels preganglionic autonomic neurons, motoneurons, and the terminal projections of select primary afferents for both light and electron microscopic studies. We demonstrate also that this method can be combined with post-embedding immunogold labeling to detect amino acid transmitters in synaptic boutons.

Different basal levels of CaMKII phosphorylated at Thr286/287 at nociceptive and low-threshold primary afferent synapses.

Postsynaptic autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286/287 is crucial for the induction of long-term potentiation at many glutamatergic synapses, and has also been implicated in the persistence of synaptic potentiation. However, the availability of CaMKII phosphorylated at Thr286/287 at individual glutamatergic synapses in vivo is unclear. We used post-embedding immunogold labelling to quantitatively analyse the ultrastructural localization of CaMKII phosphorylated at Thr286/287 (pCaMKII) at synapses formed by presumed nociceptive and low-threshold mechanosensitive primary afferent nerve endings in laminae I-IV of rat spinal cord. Immunogold labelling was enriched in the postsynaptic densities of such synapses, consistent with observations in pre-embedding immunoperoxidase-stained dorsal horn. Presynaptic axoplasm also exhibited sparse immunogold labelling, in peptidergic terminals partly associated with dense core vesicles. Analysis of single or serial pCaMKII-immunolabelled sections indicated that the large majority of synapses formed either by presumed peptidergic or non-peptidergic nociceptive primary afferent terminals in laminae I-II of the spinal cord, or by presumed low-threshold mechanosensitive primary afferent terminals in laminae IIi-IV, contained pCaMKII in their postsynaptic density. However, the postsynaptic levels of pCaMKII immunolabelling at low-threshold primary afferent synapses were only approximately 50% of those at nociceptive synapses. These results suggest that constitutively autophosphorylated CaMKII in the postsynaptic density is a common characteristic of glutamatergic synapses, thus potentially contributing to maintenance of synaptic efficacy. Furthermore, pCaMKII appears to be differentially regulated between high- and low-threshold primary afferent synapses, possibly reflecting different susceptibility to synaptic plasticity between these afferent pathways.

Lateral cervical nucleus projections to periaqueductal gray matter in cat.

The midbrain periaqueductal gray matter (PAG) integrates the basic responses necessary for survival of individuals and species. Examples are defense behaviors such as fight, flight, and freezing, but also sexual behavior, vocalization, and micturition. To control these behaviors the PAG depends on strong input from more rostrally located limbic structures, as well as from afferent input from the lower brainstem and spinal cord. Mouton and Holstege (2000, J Comp Neurol 428:389-410) showed that there exist at least five different groups of spino-PAG neurons, each of which is thought to subserve a specific function. The lateral cervical nucleus (LCN) in the upper cervical cord is not among these five groups. The LCN relays information from hair receptors and noxious information and projects strongly to the contralateral ventroposterior and posterior regions of thalamus and to intermediate and deep tectal layers. The question is whether the LCN also projects to the PAG. The present study in cat, using retrograde and anterograde tracing techniques, showed that neurons located in the lateral two-thirds of the LCN send fibers to the lateral part of the PAG, predominantly at rostrocaudal levels A0.6-P0.2. This part of the PAG is known to be involved in flight behavior. A concept is put forward according to which the LCN-PAG pathway alerts the animal about the presence of cutaneous stimuli that might represent danger, necessitating flight. J. Comp. Neurol. 471:434-445, 2004.

Glutamate, but not aspartate, is enriched in trigeminothalamic tract terminals and associated with their synaptic vesicles in the rat nucleus submedius.

To examine the possible roles of glutamate and aspartate as neurotransmitters in the nucleus submedius (Sm) of rats, the distributions of these amino acids were examined by electron microscopic immunogold labeling. High levels of glutamate were detected in trigeminothalamic tract terminals anterogradely labeled with horseradish peroxidase conjugates. These terminals also displayed a positive correlation between the densities of synaptic vesicles and gold particles signaling glutamate. In contrast, aspartate levels in such terminals were low and displayed no correlation with the density of synaptic vesicles. Terminals of presumed cortical origin contained the highest estimated levels of glutamate, but the positive correlation between glutamate signal and synaptic vesicle density did not reach statistical significance, presumably due to technical factors. The latter terminals also contained relatively high levels of aspartate, though without any correlation to synaptic vesicle density. The present findings provide strong support for glutamate, but not aspartate, as a trigeminothalamic tract neurotransmitter responsible for the fast synaptic transmission of nociceptive signals to neurons in the rat nucleus submedius. Aspartate presumably serves metabolic roles in these terminals. With respect to terminals of presumed cortical origin, our data are not at odds with the notion that also these terminals use glutamate as their neurotransmitter. Our findings do not support a neurotransmitter role for aspartate in the latter terminals, although such a role cannot be entirely refuted.

Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells.

We have explored whether gamma-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic beta-cells. To this end, beta-cells were engineered to express GABA(A)-receptor Cl(-)-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every beta-cell contains approximately 3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl(-)-currents, was observed during membrane depolarizations from -70 mV to voltages beyond -40 mV or when Ca(2+) was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca(2+) entry was inhibited using Cd(2+). Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes approximately 1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic beta-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1.

The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-beta-cyclodextrin (mbetacd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by mbetacd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by mbetacd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in mbetacd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase-polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by mbetacd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1-induced contractions were insensitive to the TRPC1 blocking antibody and to mbetacd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mbetacd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.