Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P-modulated growth hormone secretion.

Substance P is an undecapeptide affecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these effects, previous papers provided solid evidence that substance P exhibits regulatory effects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fibers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also affect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and somatostatinergic systems utilizing double label immunohistochemistry combined with high magnification light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fiber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addition, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also influences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts.

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.

Origins of endomorphin-immunoreactive fibers and terminals in different columns of the periaqueductal gray in the rat.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are endogenous ligands for mu-opioid receptors (MOR). In the central nervous system, EM-immunoreactive (IR) neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitarius (NTS). EM-IR fibers and terminals are found widely distributed in many brain areas, including the different columns of the periaqueductal gray (PAG). The hypothalamus, NTS, and PAG are closely involved in modulation of vocalization, autonomic and neuroendocrine functions, pain, and defensive behavior through endogenous opioid peptides that bind to the MOR in these regions. Projections exist from both the hypothalamus and the NTS to the PAG. In order to examine whether there are EM1- and/or EM2-ergic projections from the hypothalamus and NTS to the PAG, immunofluorescence histochemistry for EM1 and/or EM2 was combined with fluorescent retrograde tracing. In rats that had Fluoro-Gold (FG) injected into different columns of the PAG, some of the EM1- or EM2-IR neurons in the hypothalamus, but none in the NTS, were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons in the hypothalamus were distributed in the dorsomedial nucleus, areas between the dorsomedial and ventromedial nucleus, and arcuate nucleus; a few were also seen in the ventromedial, periventricular, and posterior nucleus. The present results indicate that the EM-IR fibers and terminals in the PAG originate principally from the hypothalamus. They also suggest that EMs released from hypothalamus-PAG projecting neurons might mediate or modulate various functions of the PAG through binding to the MOR.

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.

Distribution, morphology, and synaptic targets of corticothalamic terminals in the cat lateral posterior-pulvinar complex that originate from the posteromedial lateral suprasylvian cortex.

The lateral posterior (LP) nucleus is a higher order thalamic nucleus that is believed to play a key role in the transmission of visual information between cortical areas. Two types of cortical terminals have been identified in higher order nuclei, large (type II) and smaller (type I), which have been proposed to drive and modulate, respectively, the response properties of thalamic cells (Sherman and Guillery [1998] Proc. Natl. Acad. Sci. U. S. A. 95:7121-7126). The aim of this study was to assess and compare the relative contribution of driver and modulator inputs to the LP nucleus that originate from the posteromedial part of the lateral suprasylvian cortex (PMLS) and area 17. To achieve this goal, the anterograde tracers biotinylated dextran amine (BDA) or Phaseolus vulgaris leucoagglutinin (PHAL) were injected into area 17 or PMLS. Results indicate that area 17 injections preferentially labelled large terminals, whereas PMLS injections preferentially labelled small terminals. A detailed analysis of PMLS terminal morphology revealed at least four categories of terminals: small type I terminals (57%), medium-sized to large singletons (30%), large terminals in arrangements of intermediate complexity (8%), and large terminals that form arrangements resembling rosettes (5%). Ultrastructural analysis and postembedding immunocytochemical staining for gamma-aminobutyric acid (GABA) distinguished two types of labelled PMLS terminals: small profiles with round vesicles (RS profiles) that contacted mostly non-GABAergic dendrites outside of glomeruli and large profiles with round vesicles (RL profiles) that contacted non-GABAergic dendrites (55%) and GABAergic dendritic terminals (45%) in glomeruli. RL profiles likely include singleton, intermediate, and rosette terminals, although future studies are needed to establish definitively the relationship between light microscopic morphology and ultrastructural features. All terminals types appeared to be involved in reciprocal corticothalamocortical connections as a result of an intermingling of terminals labelled by anterograde transport and cells labelled by retrograde transport. In conclusion, our results indicate that the origin of the driver inputs reaching the LP nucleus is not restricted to the primary visual cortex and that extrastriate visual areas might also contribute to the basic organization of visual receptive fields of neurons in this higher order nucleus.

Presynaptic localization of an AMPA-type glutamate receptor in corticostriatal and thalamostriatal axon terminals.

The neostriatum is known to receive glutamatergic projections from the cerebral cortex and thalamic nuclei. Vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2) are located on axon terminals of corticostriatal and thalamostriatal afferents, respectively, whereas VGluT3 is found in axon terminals of cholinergic interneurons in the neostriatum. In the present study, the postsynaptic localization of ionotropic glutamate receptors was examined in rat neostriatum by the postembedding immunogold method for double labelling of VGluT and glutamate receptors. Immunoreactive gold particles for AMPA receptor subunits GluR1 and GluR2/3 were frequently found not only on postsynaptic but also on presynaptic profiles immunopositive for VGluT1 and VGluT2 in the neostriatum, and GluR4-immunoreactive particles were observed on postsynaptic and presynaptic profiles positive for VGluT1. Quantitative analysis revealed that 27-45% of GluR1-, GluR2-, GluR2/3- and GluR4-immunopositive particles found in VGluT1- or VGluT2-positive synaptic structures in the neostriatum were associated with the presynaptic profiles of VGluT-positive axons. In contrast, VGluT-positive presynaptic profiles in the neostriatum showed almost no immunoreactivity for NMDA receptor subunits NR1 or NR2A/B. Furthermore, almost no GluR2/3-immunopositive particles were observed in presynaptic profiles of VGluT3-positive (cholinergic) terminals that made asymmetric synapses in the neostriatum, or in those of VGluT1- or VGluT2-positive terminals in the neocortex. The present results indicate that AMPA receptor subunits but not NMDA receptor subunits are located on axon terminals of corticostriatal and thalamostriatal afferents, and suggest that glutamate released from these axon terminals controls the activity of the terminals through the presynaptic AMPA autoreceptors.

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Experience-dependent regulation of the zincergic innervation of visual cortex in adult monkeys.

Zinc is packaged in, and released from, a subset of glutamatergic synapses in the mammalian telencephalon where it has been shown to act as a potent neuromodulator. In order to establish the functional role for zincergic neurons in visual cortical function and plasticity we have compared the topographic distribution of zincergic terminals in the primary visual cortex (V1) of normal adult vervet monkeys (Cercopithicus aethiops) to that in monkeys monocularly deprived of visual input for short (24 h) or long (3 months) survival times. In normal animals, staining levels for zinc were highest in layers 1-3, 4b, 5 and 6 and lowest in layers 4a and 4c. The laminar and tangential patterns of zinc staining were complementary to staining patterns demonstrated using cytochrome oxidase (CO) histochemistry. Following 3 months of monocular deprivation by enucleation, levels of zinc staining in layers 3, 4calpha and 6a were heterogeneously reduced, clearly revealing the ocular dominance pattern in V1. When compared with the pattern of CO staining, levels of both CO and zinc were reduced in cortical territory innervated by the enucleated eye. Zinc histochemistry also revealed the ocular dominance pattern after only 24 h of monocular impulse blockade induced by enucleation or intravitreal tetrodotoxin infusion. However, by either means of deprivation for 24 h, levels of zinc were increased in deprived-eye stripes relative to nondeprived-eye stripes. These results indicate that zincergic terminals demarcate distinct compartments in the primate visual cortex. Furthermore, levels of synaptic zinc are rapidly and dynamically regulated, suggesting that zinc and/or zincergic neurons participate in mediating activity-dependent changes in the organization of the adult neocortex.

Honeycomb-like mosaic at the border of layers 1 and 2 in the cerebral cortex.

In this report, we present evidence of a small-scale modularity (<100 microm) at the border of layers 1 and 2 in neocortical areas. The modularity is best seen in tangential sections, with double-labeling immunohistochemistry to reveal overlapping or complementary relationships of different markers. The pattern is overall like a reticulum or mosaic but is described as a "honeycomb," in which the walls and hollows are composed of distinct afferent and dendritic systems. We demonstrate the main components of the honeycomb in rat visual cortex. These are as follows: (1) zinc-enriched, corticocortical terminations in the walls, and in the hollows, thalamocortical terminations (labeled by antibody against vesicular glutamate transporter 2 and by cytochrome oxidase); (2) parvalbumin-dense neuropil in the walls that partly colocalizes with elevated levels of glutamate receptors 2/3, NMDAR receptor 1, and calbindin; and (3) dendritic subpopulations preferentially situated within the walls (dendrites of layer 2 neurons) or hollows (dendrites of deeper neurons in layers 3 and 5). Because the micromodularity is restricted to layers 2 and 1b, without extending into layer 3, this may be another indication of a laminar-specific substructure at different spatial scales within cortical columns. The suggestion is that corticocortical and thalamocortical terminations constitute parallel circuits at the level of layer 2, where they are segregated in association with distinct dendritic systems. Results from parvalbumin staining show that the honeycomb mosaic is not limited to rat visual cortex but can be recognized at the layer 1-2 border in other areas and species.

K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis.

Two different families of Na+/Ca2+ exchangers, K+-independent NCX and K+-dependent NCKX, are known. Exploiting the outward K+ gradient, NCKX is able to extrude Ca2+ more efficiently than NCX, even when the Na+ gradient is reduced. The NCKX, which was originally thought to be limited to the retinal photoreceptor, was shown recently to be widely distributed in the brain. We investigated the contribution of Na+/Ca2+ exchange to Ca2+ clearance mechanisms in neurohypophysial (NHP) axon terminals, using patch-clamp and microfluorometry techniques. In the presence of internal K+, Ca2+ decay was significantly slowed by the removal of external Na+, indicative of the role of Na+/Ca2+ exchange. As internal [K+] was decreased, Ca2+ decay rate and its dependence on Na+ were greatly attenuated. In the absence of internal K+, Ca2+ decay rate was little affected by Na+ removal. Quantitative analysis using Ca2+ decay rate constant indicated that >60% of Ca2+ extrusion is mediated by Na+/Ca2+ exchange when peak [Ca2+] level is higher than 500 nm, and approximately 90% of Na+/Ca2+ exchange activity is K+ dependent. In situ hybridization confirmed the expression of NCKX2 transcripts in the supraoptic nucleus in which soma of NHP axon terminals are located. To our knowledge, this is the first report to show the significant role of K+-dependent Na+/Ca2+ exchange in neuronal cells other than photoreceptors. Considering that axon terminals are subject to an invasion by high-frequency Na+ spikes, which may lower Na+ gradients, the presence of NCKX may have a functional significance in intracellular Ca2+ regulation.

Thalamic distribution of zinc-rich terminal fields and neurons of origin in the rat.

Several cortico-cortical and limbic-related circuits are enriched in zinc, which is considered as an important modulator of glutamatergic transmission. While heavy metals have been detected in the thalamus, the specific presence of zinc has not been examined in this region. We have used two highly sensitive variations of the Timm method to study the zinc-rich innervation in the rat thalamus, which was compared to the distribution of acetylcholinesterase activity. The origin of some of these zinc-rich projections was also investigated by means of retrograde transport after intracerebral infusions of sodium selenium (Na2SeO3). The overall zinc staining in the thalamus was much lower than in the neocortex, striatum or basal forebrain; however, densely stained terminal fields were observed in the dorsal tip of the reticular thalamic nucleus, the anterodorsal and lateral dorsal thalamic nuclei and the zona incerta. In addition, moderately stained zinc-rich terminal fields were found in the rostral intralaminar nuclei, nucleus reuniens and lateral habenula. Intracerebral infusions of Na2SeO3 in the lateral dorsal nucleus resulted in retrogradely labeled neurons that were located in the postsubiculum, and also in the pre- and parasubiculum. These results are the first to establish the existence of a zinc-rich subicular-thalamic projection. Similar infusions in either the intralaminar nuclei or the zona incerta resulted in labeling of neurons in several brainstem structures related to the reticular formation. Our results provide morphological evidence for zinc modulation of glutamatergic inputs to highly selective thalamic nuclei, arising differentially from either cortical limbic areas or from brainstem ascending activation systems.

High vesicular monoamine transporter binding in asymptomatic bipolar I disorder: sex differences and cognitive correlates.

OBJECTIVE: It has been hypothesized that anomalies in monoaminergic function underlie some of the manifestations of bipolar disorder. In this study the authors examined the possibility that trait-related abnormalities in the concentration of monoaminergic synaptic terminals may be present in patients with asymptomatic bipolar disorder type I. METHOD: The concentration of a stable presynaptic marker, the vesicular monoamine transporter protein (VMAT2), was quantified with (+)[(11)C]dihydrotetrabenazine (DTBZ) and positron emission tomography. Sixteen asymptomatic patients with bipolar I disorder who had a prior history of mania with psychosis (nine men and seven women) and individually matched healthy subjects were studied. Correlational analyses were conducted to examine the relationship between regional VMAT2 binding, cognitive function, and clinical variables. RESULTS: VMAT2 binding in the thalamus and ventral brainstem of the bipolar patients was higher than that in the comparison subjects. VMAT2 concentrations in these regions correlated with performance on measures of frontal, executive function. In addition, sex differences in VMAT2 binding were detected in the thalamus of the bipolar patients; the male patients had higher binding than the women. No sex differences in binding were observed in the healthy comparison group. CONCLUSIONS: These initial results suggest that higher than normal VMAT2 expression and, by extension, concentration of monoaminergic synaptic terminals, may represent a trait-related abnormality in patients with bipolar I disorder and that male and female patients show different patterns. Also, VMAT2 concentrations may be associated with some of the cognitive deficits encountered in euthymic bipolar disorder.

A new class of neurotoxin from wasp venom slows inactivation of sodium current.

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX slowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Alpha-synuclein is not a requisite component of synaptic boutons in the adult human central nervous system.

It is increasingly clear that the normal protein alpha-synuclein is in some manner closely associated with presynaptic components of select neuronal types within the adult human central nervous system (CNS) and, in addition, that in its pathologically altered state alpha-synuclein aggregates selectively in the form of filamentous inclusion bodies during certain progressive neurodegenerative disorders, such as familial and sporadic Parkinson's disease. By having the antibody AFshp raised specifically to alpha-synuclein to label Parkinson disease-specific Lewy bodies and Lewy neurites as well as synaptic boutons containing the unaltered protein, an initial attempt is made to map the overall distribution pattern and describe the staining behavior of the immunoreactive punctae in select regions of the prosencephalon. Neocortical immunolabeling is most prominent in the prodigious, but incompletely myelinated, association fields and faintest in the heavily myelinated primary motor and primary sensory fields, with the premotor and first order sensory association areas occupying an intermediate position. Of the thalamic grays evaluated, those containing powerfully myelinated fiber tracts (e.g. centrum medianum, habenular complex) show the weakest immunolabeling, whereas, less sturdily myelinated structures are highly immunoreactive. The fact that the immunostaining spectrum for normal alpha-synuclein is so broad, together with the fact that some thalamic sites actually are immunonegative leads to the following conclusions (1) alpha-synuclein, although present in the synaptic boutons of many nerve cells in the adult human CNS, is by no means ubiquitous there, and (2) neuronal types lacking the normal protein cannot generate the Parkinson's disease-specific filamentous pathology.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Alterations in chandelier neuron axon terminals in the prefrontal cortex of schizophrenic subjects.

OBJECTIVE: Abnormalities in prefrontal cortical gamma-aminobutyric acid (GABA) neurotransmission may contribute to cognitive dysfunction in schizophrenia. The density of chandelier neuron axon terminals (cartridges) immunoreactive for the GABA membrane transporter (GAT-1) has been reported to be reduced in the dorsolateral prefrontal cortex of schizophrenic subjects. Because cartridges regulate the output of pyramidal cells, this study analyzed the laminar distribution of GAT-1-immunoreactive cartridges to determine whether certain subpopulations of pyramidal cells are preferentially affected. METHOD: Measurements were made of the density of GAT-1 -immunoreactive cartridges in layers 2-3a, 3b-4, and 6 of dorsolateral prefrontal cortex area 46 in 30 subjects with schizophrenia, each of whom was matched to one normal and one psychiatric comparison subject. GAT-1-immunoreactive cartridge density was also examined in monkeys chronically treated with haloperidol. RESULTS: Relative to both comparison groups, the schizophrenic subjects had significantly lower GAT-1-immunoreactive cartridge density in layers 2-3a and 3b-4. The decrease was most common and most marked in layers 3b-4, where 80% of the schizophrenic subjects exhibited an average 50.1% decrease in cartridge density in comparison with the matched normal subjects. In contrast, GAT-1-immunoreactive cartridge density was unchanged in the haloperidol-treated monkeys. CONCLUSIONS: These findings demonstrate that the density of GAT-1-immunoreactive cartridges is reduced in the majority of schizophrenic subjects and that this alteration may most prominently affect the function of pyramidal cells located in the middle cortical layers. This abnormality may reflect a number of underlying deficits, including a primary defect in dorsolateral prefrontal cortex circuitry or a secondary response to altered thalamic input to this region.

Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions.

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium-binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq-like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin- and dynamin-like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq-like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium-binding regions (EF hands) are conserved. The widespread occurrence of frq-like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons.

Co-localized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission.

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA(B) receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (-ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA(B) agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA(B) antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.

Detection of low phosphorus contents in neurofilaments of squid axons by Image-EELS contrast spectroscopy.

We show that Image-EELS is suitable for detecting relatively low phosphorus concentrations in very small axoplasmic structures of squid axons. Imaging plates and a CCD camera were used as electron sensors. From image series spanning a certain energy-loss range EELS (electron energy-loss spectra) were derived by averaging read-outs from many axoplasmic particles (APs). The ratio of these spectra to spectra of the background was plotted, showing the contrast modulation as a function of the energy loss. This new approach is called EELC (electron energy-loss-dependent contrast spectroscopy). A distinct phosphorus signal was found in APs of presynaptic terminals of the squid giant synapse, in the peripheral giant axon and, as controls, in ribosomes. Biochemical experiments supported this result. In neurofilament-enriched pellets a phosphorus signal could be directly detected by serial EELS and in electron spectroscopic micrographs. After dephosphorylation of either the pellets or the extruded axoplasm with alkaline phosphatase, phosphorus signals in electron spectroscopic micrographs were absent or much reduced in size and intensity. With Image-EELS inherent limitations of traditional element detection modes in energy filtering transmission electron microscopy can be overcome. Compared with serial EELS, the selective analysis of small areas with irregular shape is possible with greatly improved signal-to-noise ratio. The identification of the element-peak in Image-EEL spectra directly proves the presence of the element within the region of interest. For small peaks, the visualization is facilitated by the contrast presentation (EELC). However, the background subtraction modes used for elemental mapping in electron spectroscopic imaging are subject to uncertainties when elemental ionization edges like the P1,2,3 edge are examined. Imaging plates are very sensitive electron sensors with a wide dynamic range. Unlike photographic emulsions, they allow acquisition of image series covering a large energy-loss range without normalization of exposure times, and direct extraction of EEL spectra. Thus, the combination of Image-EELS and imaging plates is proposed as an efficient new tool for analytical electron microscopy.