Group I metabotropic glutamate receptors in the primate motor thalamus: subsynaptic association with cortical and sub-cortical glutamatergic afferents.

Preclinical evidence indicates that mGluR5 is a potential therapeutic target for Parkinson's disease and L-DOPA-induced dyskinesia. However, the mechanisms through which these therapeutic benefits are mediated remain poorly understood. Although the regulatory role of mGluR5 on glutamatergic transmission has been examined in various basal ganglia nuclei, very little is known about the localization and function of mGluR5 in the ventral motor and intralaminar thalamic nuclei, the main targets of basal ganglia output in mammals. Thus, we used immuno-electron microscopy to map the cellular and subcellular localization of group I mGluRs (mGluR1a and mGluR5) in the ventral motor and caudal intralaminar thalamic nuclei in rhesus monkeys. Furthermore, using double immuno-electron microscopy, we examined the subsynaptic localization of mGluR5 in relation to cortical and sub-cortical glutamatergic afferents. Four major conclusions can be drawn from these data. First, mGluR1a and mGluR5 are expressed postsynaptically on the plasma membrane of dendrites of projection neurons and GABAergic interneurons in the basal ganglia- and cerebellar-receiving regions of the ventral motor thalamus and in CM. Second, the plasma membrane-bound mGluR5 immunoreactivity is preferentially expressed perisynaptically at the edges of cortical and sub-cortical glutamatergic afferents. Third, the mGluR5 immunoreactivity is more strongly expressed in the lateral than the medial tiers of CM, suggesting a preferential association with thalamocortical over thalamostriatal neurons in the primate CM. Overall, mGluR5 is located to subserve powerful modulatory role of cortical and subcortical glutamatergic transmission in the primate ventral motor thalamus and CM.

Ultrastructure of giant thalamic terminals in the auditory cortex.

The auditory system comprises some very large axonal terminals like the endbulb and calyx of Held and "giant" corticothalamic synapses. Previously, we described a hitherto unknown population of giant thalamocortical boutons arising from the medial division of the medial geniculate body (MGm) in the Mongolian gerbil, which terminate over a wide cortical range but in a columnar manner particularly in the extragranular layers of the auditory cortex. As a first step towards an understanding of their potential functional role, we here describe their ultrastructure combining anterograde tract-tracing with biocytin and electron microscopy. Quantitative ultrastructural analyses revealed that biocytin-labelled MGm boutons reach much larger sizes than other, non-labelled boutons. Also, mitochondria occupy more space within labelled boutons whereas synapses are of similar size. Labelled boutons are very heterogeneous in size but homogeneous with respect to their ultrastructural characteristics, with asymmetric synapses containing clear, round vesicles and targeting dendritic spines. Functionally, the ultrastructure of the MGm terminals indicates that they form excitatory contacts, which may transmit their information in a rapid, powerful and high-fidelity manner onto strategically advantageous compartments of their cortical target cells.

A Sensorimotor Pathway via Higher-Order Thalamus.

We now know that sensory processing in cortex occurs not only via direct communication between primary to secondary areas, but also via their parallel cortico-thalamo-cortical (i.e., trans-thalamic) pathways. Both corticocortical and trans-thalamic pathways mainly signal through glutamatergic class 1 (driver) synapses, which have robust and efficient synaptic dynamics suited for the transfer of information such as receptive field properties, suggesting the importance of class 1 synapses in feedforward, hierarchical processing. However, such a parallel arrangement has only been identified in sensory cortical areas: visual, somatosensory, and auditory. To test the generality of trans-thalamic pathways, we sought to establish its presence beyond purely sensory cortices to determine whether there is a trans-thalamic pathway parallel to the established primary somatosensory (S1) to primary motor (M1) pathway. We used trans-synaptic viral tracing, optogenetics in slice preparations, and bouton size analysis in the mouse (both sexes) to document that a circuit exists from layer 5 of S1 through the posterior medial nucleus of the thalamus to M1 with glutamatergic class 1 properties. This represents a hitherto unknown, robust sensorimotor linkage and suggests that the arrangement of parallel direct and trans-thalamic corticocortical circuits may be present as a general feature of cortical functioning.SIGNIFICANCE STATEMENT During sensory processing, feedforward pathways carry information such as receptive field properties via glutamatergic class 1 synapses, which have robust and efficient synaptic dynamics. As expected, class 1 synapses subserve the feedforward projection from primary to secondary sensory cortex, but also a route through specific higher-order thalamic nuclei, creating a parallel feedforward trans-thalamic pathway. We now extend the concept of cortical areas being connected via parallel, direct, and trans-thalamic circuits from purely sensory cortices to a sensorimotor cortical circuit (i.e., primary sensory cortex to primary motor cortex). This suggests a generalized arrangement for corticocortical communication.

Major Feedforward Thalamic Input Into Layer 4C of Primary Visual Cortex in Primate.

One of the underlying principles of how mammalian circuits are constructed is the relative influence of feedforward to recurrent synaptic drive. It has been dogma in sensory systems that the thalamic feedforward input is relatively weak and that there is a large amplification of the input signal by recurrent feedback. Here we show that in trichromatic primates there is a major feedforward input to layer 4C of primary visual cortex. Using a combination of 3D-electron-microscopy and 3D-confocal imaging of thalamic boutons we found that the average feedforward contribution was about 20% of the total excitatory input in the parvocellular (P) pathway, about 3 times the currently accepted values for primates. In the magnocellular (M) pathway it was around 15%, nearly twice the currently accepted values. New methods showed the total synaptic and cell densities were as much as 150% of currently accepted values. The new estimates of contributions of feedforward synaptic inputs into visual cortex call for a major revision of the design of the canonical cortical circuit.

Striatal Reinnervation Process after Acute Methamphetamine-Induced Dopaminergic Degeneration in Mice.

Methamphetamine (METH), an amphetamine derivate, may increase the risk of developing Parkinson's disease (PD). Human and animal studies have shown that METH produces persistent dopaminergic neurotoxicity in the nigrostriatal pathway, despite initial partial recovery. To determine the processes leading to early compensation, we studied the detailed morphology and distribution of tyrosine hydroxylase immunoreactive fibers (TH-ir) classified by their thickness (types I-IV) before and after METH. Applying three established neurotoxic regimens of METH: single high dose (1 × 30 mg/kg), multiple lower doses (3 × 5 mg/kg) or (3 × 10 mg/kg), we show that METH primarily damages type I fibers (the thinner ones), and to a much lesser extend types II-IV fibers including sterile axons. The striatal TH terminal partial recovery process, consisting of a progressive regrowth increases in types II, III, and IV fibers, demonstrated by co-localization of GAP-43, a sprouting marker, was observed 3 days post-METH treatment. In addition, we demonstrate the presence of growth-cone-like TH-ir structures, indicative of new terminal generation as well as improvement in motor functions after 3 days. A temporal relationship was observed between decreases in TH-expression and increases in silver staining, a marker of degeneration. Striatal regeneration was associated with an increase in astroglia and decrease in microglia expression, suggesting a possible role for the neuroimmune system in regenerative processes. Identification of regenerative compensatory mechanisms in response to neurotoxic agents could point to novel mechanisms in countering the neurotoxicity and/or enhancing the regenerative processes.

Single excitatory axons form clustered synapses onto CA1 pyramidal cell dendrites.

CA1 pyramidal neurons are a major output of the hippocampus and encode features of experience that constitute episodic memories. Feature-selective firing of these neurons results from the dendritic integration of inputs from multiple brain regions. While it is known that synchronous activation of spatially clustered inputs can contribute to firing through the generation of dendritic spikes, there is no established mechanism for spatiotemporal synaptic clustering. Here we show that single presynaptic axons form multiple, spatially clustered inputs onto the distal, but not proximal, dendrites of CA1 pyramidal neurons. These compound connections exhibit ultrastructural features indicative of strong synapses and occur much more commonly in entorhinal than in thalamic afferents. Computational simulations revealed that compound connections depolarize dendrites in a biophysically efficient manner, owing to their inherent spatiotemporal clustering. Our results suggest that distinct afferent projections use different connectivity motifs that differentially contribute to dendritic integration.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

An Ultrastructural Study of the Thalamic Input to Layer 4 of Primary Motor and Primary Somatosensory Cortex in the Mouse.

The traditional classification of primary motor cortex (M1) as an agranular area has been challenged recently when a functional layer 4 (L4) was reported in M1. L4 is the principal target for thalamic input in sensory areas, which raises the question of how thalamocortical synapses formed in M1 in the mouse compare with those in neighboring sensory cortex (S1). We identified thalamic boutons by their immunoreactivity for the vesicular glutamate transporter 2 (VGluT2) and performed unbiased disector counts from electron micrographs. We discovered that the thalamus contributed proportionately only half as many synapses to the local circuitry of L4 in M1 compared with S1. Furthermore, thalamic boutons in M1 targeted spiny dendrites exclusively, whereas ∼9% of synapses were formed with dendrites of smooth neurons in S1. VGluT2+ boutons in M1 were smaller and formed fewer synapses per bouton on average (1.3 vs 2.1) than those in S1, but VGluT2+ synapses in M1 were larger than in S1 (median postsynaptic density areas of 0.064 µm2 vs 0.042 µm2). In M1 and S1, thalamic synapses formed only a small fraction (12.1% and 17.2%, respectively) of all of the asymmetric synapses in L4. The functional role of the thalamic input to L4 in M1 has largely been neglected, but our data suggest that, as in S1, the thalamic input is amplified by the recurrent excitatory connections of the L4 circuits. The lack of direct thalamic input to inhibitory neurons in M1 may indicate temporal differences in the inhibitory gating in L4 of M1 versus S1.SIGNIFICANCE STATEMENT Classical interpretations of the function of primary motor cortex (M1) emphasize its lack of the granular layer 4 (L4) typical of sensory cortices. However, we show here that, like sensory cortex (S1), mouse M1 also has the canonical circuit motif of a core thalamic input to the middle cortical layer and that thalamocortical synapses form a small fraction (M1: 12%; S1: 17%) of all asymmetric synapses in L4 of both areas. Amplification of thalamic input by recurrent local circuits is thus likely to be a significant mechanism in both areas. Unlike M1, where thalamocortical boutons typically form a single synapse, thalamocortical boutons in S1 usually formed multiple synapses, which means they can be identified with high probability in the electron microscope without specific labeling.

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction.

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(-)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(-) NMJs was significantly diminished, and that new boutons in Syn(-) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(-) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.

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.

Synaptic and extrasynaptic location of the receptor tyrosine kinase met during postnatal development in the mouse neocortex and hippocampus.

MET, a replicated autism risk gene, encodes a pleiotropic receptor tyrosine kinase implicated in multiple cellular processes during development and following injury. Previous studies suggest that Met modulates excitatory synapse development in the neocortex and hippocampus, although the underlying mechanism is unknown. The peak of Met expression corresponds to the period of process outgrowth and synaptogenesis, with robust expression in hippocampal and neocortical neuropil. Resolving whether neuropil expression represents presynaptic, postsynaptic or glial localization provides insight into potential mechanisms of Met action. The subcellular distribution of Met was characterized using complementary ultrastructural, in situ proximity ligation assay (PLA), and biochemical approaches. At postnatal day (P) 7, immunoelectron microscopy revealed near-equivalent proportions of Met-immunoreactive pre- (axons and terminals) and postsynaptic (dendritic shafts and spines) profiles in the stratum radiatum in the hippocampal CA1 region. Staining was typically in elements in which the corresponding pre- or postsynaptic apposition was unlabeled. By P21, Met-immunoreactive presynaptic profiles predominated and ~20% of Met-expressing profiles were glial. A different distribution of Met-immunoreactive profiles was observed in layer V of somatosensory cortex: Met-labeled spines were rare and a smaller proportion of glial profiles expressed Met. Strikingly, Met-immunoreactive presynaptic profiles predominated over postsynaptic profiles as early as P7. PLA analysis of neurons in vitro and biochemical analysis of tissue subsynaptic fractions confirmed the localization of Met in specific synaptic subcompartments. The study demonstrates that Met is enriched at synapses during development and its activation may modulate synapse formation and stability through both pre- and postsynaptic mechanisms.

Terminals of the major thalamic input to visual cortex are devoid of synapsin proteins.

Synapsins are nerve-terminal proteins that are linked to synaptic transmission and key factors in several forms of synaptic plasticity. While synapsins are generally assumed to be ubiquitous in synaptic terminals, whether they are excluded from certain types of terminals is of interest. In the visual pathway, synapsins are lacking in photoreceptor and bipolar cell terminals as well as in retinogeniculate synapses. These are the terminals of the first three feedforward synapses in the visual pathway, implying that lack of synapsins may be a common property of terminals that provide the primary driver activity onto their postsynaptic neurons. To further investigate this idea, we studied the fourth driver synapse, thalamocortical synapses in visual cortex, using glutamatergic terminal antibody markers anti-VGluT1 and VGluT2, anti-Synapsin I and II, and confocal microscopy to analyze co-localization of these proteins in terminals. We also used pre-embedding immunocytochemical labeling followed by electron microscopy to investigate morphological similarities or differences between terminals containing synapsins or VGluT2. In visual cortex, synapsin coincided extensively with non-TC-neuron marker, VGluT1, while thalamocortical terminal marker VGluT2 and synapsin overlap was sparse. Morphologically, synapsin-stained terminals were smaller than non-stained, while VGluT2-positive thalamocortical terminals constituted the largest terminals in cortex. The size discrepancy between synapsin- and VGluT2-positive terminals, together with the complementary staining patterns, indicates that thalamocortical synapses are devoid of synapsins, and support the hypothesis that afferent sensory information is consistently transmitted without the involvement of synapsins. Furthermore, VGluT2 and synapsins were colocalized in other brain structures, suggesting that lack of synapsins is not a property of VGluT2-containing terminals, but a property of primary driver terminals in the visual system.

The impact of adolescent social isolation on dopamine D2 and cannabinoid CB1 receptors in the adult rat prefrontal cortex.

Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior.

Synaptic organization of the rat thalamus: a quantitative study.

First-order thalamic nuclei receive driving afferents from ascending pathways and transmit processed information to the cortex. Higher-order thalamic nuclei receive driver messages from layer 5 of cortex and transmit information from one cortical area to the other. The different types of axon terminals RL (round vesicles, large terminals), RS (round vesicles, small terminals) and F (flattened vesicles) and their synaptic junctions have been here compared in three first-order (ventrobasal, lateral geniculate and anteroventral) and three higher-order (posterior, lateral posterior and mediodorsal) thalamic nuclei of the rat. In the present study, the higher-order relays differ from first-order relays as in the cat, in having fewer driver terminals (RL) and synapses than do the first-order relays. However, the F terminals showed opposite ratios in the first versus higher-order thalamic nuclei. The majority of the terminals in all thalamic nuclei studied were RS terminals. The area measurements of the three types of terminals and synaptic lengths showed no significant differences between first and higher-order nuclei. The driver inputs represent the minority and the modulatory inputs represent the majority of the terminals and synapses in all thalamic nuclei. In conclusion, there is a relative paucity of driver inputs, whereas modulatory inputs establish more numerous synapses to achieve finer modulation.

Distribution of large terminal inputs from the primary and secondary somatosensory cortices to the dorsal thalamus in the rodent.

The present study was undertaken to determine the precise projection pattern from the primary (S1) and secondary (S2) somatosensory cortices to the posterior nuclear proper (POm) and ventroposterior thalamic nuclei (VP). The POm was previously shown to receive large boutons arising exclusively from layer V of the S1 barrel region. This descending input was proposed to play a key role, namely, as a driver, in shaping the receptive property of POm neurons. To determine whether other body parts and the S2 also contribute such unique inputs to the dorsal thalamus, anterograde neuroanatomical tracers were focally deposited in the S1 and S2 forepaw and whisker regions of rats and C57BL6-Tg (GFPm)/Thy1 transgenic mice. Our major findings were that, 1) irrespective of body representations, both the S1 and the S2 provided corticothalamic large terminals to the POm with comparable morphological characteristics and 2) descending large terminals were also noted in particular subzones within the VP, including boundary and caudal areas. We concluded, based on these findings, that the rodent VP has three partitions: the rostral VP innervated by small corticothalamic terminals, the caudal VP with both corticothalamic small and large terminals, and a surrounding shell region, which also contained large terminals. Furthermore, assuming that the large terminal has a driver's role, we propose that particular subzones in the VP may play a role as a multiple-order thalamic relay so that they can simultaneously coordinate with first- and higher-order relays in the thalamocortical circuitry for processing somatosensory information.

Carboxypeptidase E cytoplasmic tail mediates localization of synaptic vesicles to the pre-active zone in hypothalamic pre-synaptic terminals.

How synaptic vesicles (SVs) are localized to the pre-active zone (5-200 nm beneath the active zone) in the nerve terminal, which may represent the slow response SV pool, is not fully understood. Electron microscopy revealed the number of SVs located in the pre-active zone, was significantly decreased in hypothalamic neurons of carboxypeptidase E knockout (CPE-KO) mice compared with wild-type mice. Additionally, we found K(+)-stimulated glutamate secretion from hypothalamic embryonic neurons was impaired in CPE-KO mice. Biochemical studies indicate that SVs from the hypothalamus of wild-type mice and synaptic-like microvesicles from PC12 cells contain a transmembrane form of CPE, with a cytoplasmic tail (CPE(C10)), maybe involved in synaptic function. Yeast two-hybrid and pull-down experiments showed that the CPE cytoplasmic tail interacted with gamma-adducin, which binds actin enriched at the nerve terminal. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that expression of GFP-CPE(C15) reduced the steady-state level of synaptophysin-mRFP containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings identify the CPE cytoplasmic tail, as a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells.

Distribution of the SNAP25 and SNAP23 synaptosomal-associated protein isoforms in rat cerebellar cortex.

Synaptosome-associated protein of 25 kDa (SNAP25) is a component of the fusion complex that mediates synaptic vesicle exocytosis, regulates calcium dynamics and neuronal plasticity. Despite its crucial role in vesicle release, SNAP25 is not distributed homogenously within the brain. It seems to be virtually absent in mature inhibitory terminals and is observed in a subtype of excitatory neurons defined by the expression of vesicular glutamate transporter 1 (VGluT1). Since a complementary distribution of VGluT1 and VGluT2 in excitatory synapses is correlated with different probabilities of release (Pr), we evaluated whether SNAP25 localization is associated with specific synaptic properties. In the cerebellum, climbing fiber (CF) and parallel fiber (PF) inputs, which impinge onto the same Purkinje cell (PC), have very different functional properties. In the cerebellum of adult rats, using confocal and electron microscopy, we observed that VGluT2-positive CFs, characterized by a high Pr, only weakly express SNAP25, while VGluT1-positive PFs that show a low Pr abundantly express SNAP25. Moreover, SNAP25 was less profuse in the VGluT2-positive rosettes of mossy fibers (MFs) and was almost absent in inhibitory terminals. We extended our analysis to the SNAP23 homolog; this is expressed at different levels in both gamma-aminobutyric acid-containing terminals (GABAergic) and glutamatergic terminals of the cerebellar cortex. In conclusion, the preferential localization of SNAP25 in specific synaptic boutons suggests a correlation between SNAP25 and the Pr. This evidence supports the hypothesis that SNAP25 has a modulatory role in shaping synaptic responses.

Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging.

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode.

On the nature of the intrinsic connectivity of the cat motor cortex: evidence for a recurrent neural network topology.

The details and functional significance of the intrinsic horizontal connections between neurons in the motor cortex (MCx) remain to be clarified. To further elucidate the nature of this intracortical connectivity pattern, experiments were done on the MCx of three cats. The anterograde tracer biocytin was ejected iontophoretically in layers II, III, and V. Some 30-50 neurons within a radius of approximately 250 microm were thus stained. The functional output of the motor cortical point at which biocytin was injected, and of the surrounding points, was identified by microstimulation and electromyographic recordings. The axonal arborizations of the stained neurons were traced under camera lucida. The axon collaterals were extensive, reaching distances of

Catecholaminergic input to the oxytocin neurosecretory system in the human hypothalamus.

Previous studies revealed that oxytocin release is increased by various forms of stress. Hypertonic saline injection, immobilization, and several other stressors elevated the blood level of oxytocin in rats. However, the mechanism of the stress-induced oxytocin release in human is not elucidated yet. Although numerous studies indicate that catecholamines play a pivotal role in modulating the release of oxytocin, there is a lack of data regarding the morphological substrate of this phenomenon. In order to reveal putative juxtapositions between tyrosine hydroxylase-immunoreactive (TH-IR) catecholaminergic and the oxytocinergic systems in the human hypothalamus, we utilized double-label immunohistochemistry in the present study. Numerous TH-IR axon varicosities abutted on oxytocin-IR neurons in the supraoptic and paraventricular nuclei, forming synapse-like associations. Close examination of these juxtapositions with high magnification failed to reveal any gaps between the contacting elements. In summary, the intimate associations between the TH-IR and oxytocin-IR elements may be functional synapses and may represent the morphological substrate of stress-influenced oxytocin release. The finding that several oxytocin-IR perikarya did not receive apparent TH innervation suggests that additional mechanisms may play significant roles in the oxytocin modulation by stressors.