SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells.

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK(1) cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15.

Hrs-2, via interactions with SNAP-25, plays a regulatory role on the exocytic machinery. We now show that Hrs-2 physically interacts with Eps15, a protein required for receptor-mediated endocytosis. The Hrs-2/Eps15 interaction is calcium dependent, inhibited by SNAP-25 and alpha-adaptin, and results in the inhibition of receptor-mediated endocytosis. Immunoelectron microscopy reveals Hrs-2 localization on the limiting membrane of multivesicular bodies, organelles in the endosomal pathway. These data show that Hrs-2 regulates endocytosis, delineate a biochemical pathway (Hrs-2-Eps15-AP2) in which Hrs-2 functions, and suggest that Hrs-2 acts to provide communication between endo- and exocytic processes.

Distinct protein domains are responsible for the interaction of Hrs-2 with SNAP-25. The role of Hrs-2 in 7 S complex formation.

Regulated secretion of neurotransmitter at the synapse is likely to be mediated by dynamic protein interactions involving components of the vesicle (vesicle-associated membrane protein; VAMP) and plasma membrane (syntaxin and synaptosomal associated protein of 25 kDa (SNAP-25)) along with additional molecules that allow for the regulation of this process. Recombinant Hrs-2 interacts with SNAP-25 in a calcium-dependent manner (they dissociate at elevated calcium levels) and inhibits neurotransmitter release. Thus, Hrs-2 has been hypothesized to serve a negative regulatory role in secretion through its interaction with SNAP-25. In this report, we show that Hrs-2 and SNAP-25 interact directly through specific coiled-coil domains in each protein. The presence of syntaxin enhances the binding of Hrs-2 to SNAP-25. Moreover, while both Hrs-2 and VAMP can separately bind to SNAP-25, they cannot bind simultaneously. Additionally, the presence of Hrs-2 reduces the incorporation of VAMP into the syntaxin.SNAP-25.VAMP (7 S) complex. These findings suggest that Hrs-2 may modulate exocytosis by regulating the assembly of a protein complex implicated in membrane fusion.

The cellular and developmental expression of hrs-2 in rat.

The molecular events underlying vesicular trafficking probably involve the formation and dissolution of protein complexes between integral components of the vesicle and its target membrane. SNAP-25 is associated with the plasma membrane and is a component of a core protein complex thought to be essential for neurotransmitter release. We have previously characterized a protein, hrs-2, that interacts with SNAP-25 and inhibits secretion from permeabilized PC12 cells. The cellular localization and developmental expression patterns of a number of proteins involved in the secretion machinery have been documented. To understand more about the possible cellular role of hrs-2, we have examined hrs-2 distribution, developmental expression and subcellular localization in rat tissues and cell lines. We show herein that the distribution of hrs-2 in brain and periphery parallels that of SNAP-23/25, and that recombinant hrs-2 binds to both SNAP-23 and SNAP-25. Hrs-2 mRNA and protein are found almost ubiquitously in neurons in the brain. Hrs-2 mRNA is expressed in the neural tube at E10 and thereafter mRNA and protein levels remain relatively constant in the whole brain through adulthood. In cultured PC12 cells, endogenous hrs-2 is expressed in the cytoplasm and on the limiting membranes of multivesicular bodies. Overexpression of hrs-2 in mammalian cells results in the appearance of large intracellular compartments that are labelled with hrs-2 antibodies. The wide distribution, the interaction with SNAP-23 and the localization on multivesicular body membranes suggest a general role for hrs-2 in cellular machinery.

Isoform-specific exocytotic protein mRNA expression in hypothalamic magnocellular neurons: regulation after osmotic challenge.

Exocytosis is regulated by proteins which interact to promote docking and fusion of secretory granules with the plasma membrane. We have used in situ hybridization to study the mRNA expression for vesicle-associated membrane protein (VAMP) isoforms VAMP-1 and VAMP-2, synaptosomal-associated protein of 25-kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, mammalian homologue of unc-18 (munc-18) and Hrs-2 in neurosecretory neurons of the magnocellular paraventricular (PVN) and supraoptic (SON) nuclei of normal and osmotically challenged animals. In PVN and SON neurons of normal animals, strong labeling was demonstrated for VAMP-2 and SNAP-25a mRNA, whereas VAMP-1 or SNAP-25b mRNA could not be detected. Salt-loading (2% NaCl as drinking water), an animal model which increases the expression and secretion of hormones from hypothalamic magnocellular neurons, resulted in significantly increased mRNA levels for VAMP-2 (36%, 28%), munc-18 (74%, 68%) and SNAP-25a (59%, 77%) in the PVN and SON, respectively. There was no significant increase in Hrs-2 mRNA levels in the PVN, whereas a significant increase (22%) was observed in the SON. In the posterior pituitary, immunohistochemistry showed a marked decrease in numbers and intensity of vasopressin-immunoreactive (-IR) nerve endings after salt-loading. There were no obvious changes in numbers or intensity of VAMP-2-, munc-18-, Hrs-2- or SNAP-25-IR fibers. Large varicosities containing VAMP-2- and Hrs-2 immunocreactivity were seen in salt-loaded animals. The results show isoform-specific mRNA expression in neurosecretory neurons and an increased mRNA expression of proteins participating in the molecular regulation of exocytosis during an experimental situation characterized by increased secretion.

Hrs-2 is an ATPase implicated in calcium-regulated secretion.

Associations between proteins present on neurotransmitter-containing vesicles and on the presynaptic membrane are thought to underlie docking and fusion of synaptic vesicles with the plasma membrane, which are obligate steps in regulated neurotransmission. SNAP-25 resides on the plasma membrane and interacts with syntaxin (a plasma membrane t-SNARE) and VAMP (a vesicle v-SNARE) to form a core protein complex thought to be an intermediate in a biochemical pathway that is essential for vesicular transport. We have now characterized a protein, Hrs-2, that interacts with SNAP-25. The binding of Hrs-2 to SNAP-25 is inhibited by calcium in the physiological concentration range that supports synaptic transmission. Furthermore, Hrs-2 binds and hydrolyses nucleoside triphosphates with kinetics that suggest that ATP is the physiological substrate for this enzyme. Hrs-2 is expressed throughout the brain and is present in nerve terminals. Moreover, recombinant Hrs-2 inhibits calcium-triggered 3H-noradrenaline release from permeabilized PC12 cells. Our results suggest a role for Hrs-2 in regulating secretory processes through calcium- and nucleotide-dependent modulation of vesicle-trafficking protein complexes.

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord--IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/peptide histidine isoleucine.

Using immunofluorescence histochemistry and pre- and post-embedding immunoelectron microscopy the rat lumbar dorsal horn was analysed in normal rats and 14 days after unilateral transection of the sciatic nerve. A marked increase in neuropeptide Y-like immunoreactivity was observed in the ipsilateral, superficial dorsal horn, especially in laminae III and IV, of the lumbar 4-5 spinal cord segments after peripheral axotomy. In the ipsilateral lamina II two types of neuropeptide Y-immunoreactive, presumably primary afferent terminals could be identified at the ultrastructural level. The first type contained many large dense-core vesicles (100-155 nm in diameter), whereas a second, more common type had only a few and smaller large dense-core vesicles (80-100 nm in diameter), plus synaptic vesicles of varying diameter (50-85 nm), large empty vesicles and tubular structures. Only occasionally were neuropeptide Y-positive terminals in lamina II involved in the formation of axonal labyrinths. In the ipsilateral lamina III, the number of neuropeptide Y-positive nerve terminals markedly increased after axotomy, with a moderate increase in lamina IV. These neuropeptide Y-positive terminals were morphologically similar to the second type of neuropeptide Y-positive terminal in lamina II, i.e. contained many synaptic vesicles (45-50 nm in diameter), a few small large dense-core vesicles (80-100 nm in diameter), electron-dense granular matrix and a few tubular structures. Fusion of synaptic vesicles with the plasma membrane was often observed at these synapses. These terminals frequently formed glomeruli but were not involved in axonal labyrinths. With regard to local neurons, neuropeptide Y-like immunoreactivity was observed in many dendrite-like profiles mostly making synaptic contacts with neuropeptide Y-negative dendrites and only rarely contacting the central terminal of the glomeruli. Neuropeptide Y-positive nerve endings were mainly seen in lamina I and the outer third of lamina II. After peripheral axotomy the number of vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive terminals was increased in laminae I and II. They contained many large dense-core vesicles (100-120 nm in diameter), and some of them were positive for vasoactive intestinal polypeptide/peptide histidine isoleucine. Morphologically, the terminals were characterized by a granular matrix, tubular structures, empty vesicles, reduction in synaptic vesicles and absence of postsynaptic densities. Vasoactive intestinal polypeptide/peptide histidine isoleucine-like immunoreactivities were often found in association with labyrinth formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord--III. Effects of peripheral axotomy with special reference to galanin.

In this study co-localization of galanin- with calcitonin gene-related peptide (CGRP)-like immunoreactivity was examined in dorsal root ganglion neurons 14 days after sciatic nerve cut using a laser scanning confocal microscope. CGRP- and galanin-like immunoreactivities were also analysed in the dorsal horn of the spinal cord of these animals with immunofluorescence microscopy. The ultrastructural changes in galanin-immunoreactive, presumably primary afferent terminals in the superficial dorsal horn, were studied as well as the relationship between galanin-, substance P- and CGRP-like immunoreactivities in primary afferent terminals. Local galanin-positive neurons in lamina II were also analysed after peripheral axotomy. Under the confocal microscope, CGRP-like immunoreactivity was located in the perinuclear region, probably the Golgi complex, and in dot-like structures, probably representing large dense-core vesicles, in normal dorsal root ganglion neurons. However, after peripheral axotomy CGRP was mainly detected in dot-like structures. Only a slight decrease in percentage of CGRP neurons in dorsal root ganglion was seen after axotomy, and about 84% of the galanin-positive neurons contained CGRP. The field of galanin-positive nerve fibres in the superficial lumbar (L)4 and L5 dorsal horn expanded and the intensity of staining for CGRP was reduced in these regions 14 days after sciatic nerve cut. Using pre-embedding immunoelectron microscopy, several morphological changes were observed in galanin-positive terminals in laminae I and II ipsilateral to the lesion. Most importantly, the most frequently occurring type of galanin-positive terminals (type 1) showed distinct changes with a granular matrix, many immunoreactive, peripherally located large dense-core vesicles, empty large vesicles and synaptic vesicles which were displaced from the presynaptic zone. Other galanin-positive terminals underwent even more pronounced morphological changes, including extensive vesiculolysis, also of large dense-core vesicles, filamentous degeneration or formation of axonal labyrinths. An increased number of galanin-positive nerve terminals was observed in lamina III of the ipsilateral dorsal horn after axotomy. They did not form glomeruli and contained few large dense-core vesicles. Post-embedding immunocytochemistry combined with quantitative analysis revealed that significant changes occurred in a proportion of terminals also with regard to peptide content in large dense-core vesicles after axotomy. Thus, the percentage of galanin-positive large dense-core vesicles increased in several cases and that of substance P- and CGRP-immunoreactive ones decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells.

Several proteins that are of importance for membrane trafficking in the nerve terminal have recently been characterized. We have used Western blot and immunohistochemistry to show that synaptotagmin, synaptobrevin/VAMP (vesicle-associated membrane protein), SNAP-25 (synaptosomal-associated protein of 25 kDa), and syntaxin proteins are present in cells of the islets of Langerhans in the endocrine pancreas. Synaptotagmin-like immunoreactivity (-LI) was localized to granules within the cytoplasm of a few endocrine cells located in the periphery of the islets, identified as somatostatin-containing cells, and in many nerve fibers within the islets. VAMP-LI was seen in granules of virtually all pancreatic islet cells and also in nerve fibers. SNAP-25-LI and syntaxin-LI were predominantly present in the plasma membrane of the endocrine cells, including insulin-producing beta cells. In situ hybridization, using isoform-specific oligonucleotide probes, detected VAMP-2, cellubrevin, SNAP-25, syntaxin 1A, 4, and 5, and munc-18 mRNAs in isolated pancreatic islets and in insulin-producing cells. The results show the presence of several synaptic proteins at protein and mRNA levels in pancreatic islet cells, suggesting that they may have specific roles in the molecular regulation of exocytosis also in insulin-secreting cells.

Peptide secretion: what do we know?

Understanding factors that regulate peptide release became an issue when the presence and possible role of these compounds as transmitter/modulators in various systems were realized. Many studies measuring the levels of peptides in various tissues and fluids have been performed using radioimmunoassay. However, because these peptides are measured in postmortem tissues, or perfusates that are collected at time intervals that do not approach the time scale used for exocytosis, limited information can be derived from these data. Recently the quantitative use of Northern analysis, RNase protection assays, and in situ hybridization has led to a large literature reporting on changes in peptide mRNA levels as a consequence of a variety of treatments. The assumptions involved in using radioimmunoassay measurements of peptide levels and the various methods used to measure peptide mRNAs are different, but data obtained from experiments using both methods are nonetheless used as an indication of the regulation of peptidergic neurons, and ultimately of peptide release. The mechanisms dedicated to translating cellular input into alterations in secretion have begun to be appreciated at a molecular level. Herein we will discuss the cell biology of regulated secretion and consider some levels in this pathway at which peptide release may be controlled.

Regulation of brain-derived neurotrophic factor (BDNF) expression and release from hippocampal neurons is mediated by non-NMDA type glutamate receptors.

We have examined the influence of glutamate on cortical brain-derived neurotrophic factor (BDNF) expression using in situ hybridization and immunohistochemistry. Kainic acid (KA) produced an upregulation of hippocampal and neocortical BDNF mRNA as well as BDNF protein that was blocked by a non-NMDA antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but was not affected by the NMDA antagonist 2-amino-7-phosphonoheptanoic acid (AP7). Basal levels of BDNF mRNA were not affected by NMDA, DNQX, or AP7 treatment. BDNF protein was also increased after kainate exposure with a spatial and temporal course distinct from that seen for the expression of BDNF mRNA. A dramatic shift in BDNF immunoreactivity (-IR) was observed from intracellular compartments to the neuropil surrounding CA3 pyramidal cells 2-3 hr after KA exposure. This shift in localization of BDNF-IR suggests a constitutive release of BDNF at the level of the cell body and dendrites. Moreover, we have localized mRNAs for full-length and truncated trkB, to a co-incident population of neurons and glia. These data suggest the neurons that produce BDNF also express components necessary for a biological response to the same neurotrophic factor. The present study also demonstrates increased BDNF-IR in the mossy fiber terminal zone of hippocampus after exposure to KA, as well as an increase in trkB mRNA, and provides evidence of local release of this neurotrophin into the surrounding neuropil where it would be available for local utilization. The synthesis and putative release of BDNF from somatic and/or dendritic sites within the hippocampus provide evidence of a potential autocrine or paracrine role for BDNF, and establish a local source of trophic support for the maintenance of synaptic plasticity and anatomic reorganization in the mature nervous system.

Catechol-O-methyltransferase mRNA in the kidney and its appearance during ontogeny.

Catechol-O-methyltransferase (COMT), primarily present as a soluble cytosolic form (S-COMT), inactivates catechols. The recent cloning of the rat and human S-COMT from placenta has allowed us to synthesize complementary oligonucleotide probes to study the localization of COMT mRNA during development in the rat kidney and in the adult human kidney using in situ hybridization histochemistry. In the adult rat kidney, COMT mRNA was detected in segment S3 of proximal tubule cells in the outer stripe of the outer medulla, and thick ascending limb of loop of Henle (TAL) in the inner stripe. COMT mRNA was detected in the prenatal rat kidney as early as on day 18. In the human kidney, strong hybridization signal was seen in the medulla and in tubule segments of the cortex. In the adult rat kidney, COMT mRNA was in addition demonstrated in the transitional epithelium of the ureter. The results suggest synthesis of COMT and inactivation of catechols along the distal parts of proximal tubules, in TAL cells, and in the epithelium of the ureter.

Production of Labeled cRNA Probes without Vector Cloning: Application to Localization of Basic Fibroblast Growth Factor and Tyrosine Hydroxylase mRNAs by in Situ Hybridization Histochemistry.

We describe an approach for generating labeled, single-stranded cRNA probes, using the polymerase chain reaction with primers containing RNA polymerase promoter sequences. Transcription reactions using the amplified products and RNA polymerases yield, for the most part, full-length products. cRNA probes for basic fibroblast growth factor and tyrosine hydroxylase which have incorporated (35) S-labeled nucleotides were used successfully for in situ hybridization histochemistry. This method provides a significantly faster, less work-intensive procedure for obtaining labeled single-stranded RNA useful for nucleic acid hybridization studies.

Differential expression of acidic and basic FGF in the rat substantia nigra during development.

Both acidic (aFGF) and basic (bFGF) fibroblast growth factors have been shown to be present in the adult rat ventral mesencephalon and to exert effects on cultured mesencephalic cells. In the present study we have examined the expression of aFGF and bFGF in the rat ventral mesencephalon at various stages of development. bFGF was present at all ages examined [embryonic day 16 (E16) to postnatal day 90 (P90)]. In contrast, aFGF was not detectable at embryonic and early postnatal ages, but was observed at later (P20, P60, P90) postnatal stages. These data suggest that aFGF and bFGF may have functions in mesencephalic dopamine neurones in different stages of development.

Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls.

A human genomic clone encompassing exons 1-3 of the neurotensin/neuromedin N gene was identified using a canine neurotensin complementary DNA probe. Sequence comparisons revealed that the 120-amino acid portion of the precursor sequence encoded by exons 1-3 is 89% identical to previously determined cow and dog sequences and that the proximal 250 bp of 5' flanking sequences are strikingly conserved between rat and human. The 5' flanking sequence contains cis-regulatory sites required for the induction of neurotensin/neuromedin N gene expression in PC12 cells, including AP1 sites and two cyclic adenosine-5'-monophosphate response elements. Oligonucleotide probes based on the human sequence were used to examine the distribution of neurotensin/neuromedin N messenger RNA in the ventral mesencephalon of schizophrenics and age- and sex-matched controls. Neurotensin/neuromedin N messenger RNA was observed in ventral mesencephalic cells some of which also contained melanin pigment or tyrosine hydroxylase messenger RNA. Neurons expressing neurotensin/neuromedin N messenger RNA were observed in the ventral mesencephalon of both schizophrenic and non-schizophrenic humans.

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.

Low levels of somatostatin-like immunoreactivity in neocortex resected from presumed seizure foci in epileptic patients.

The concentration of somatostatin-like immunoreactivity (SS-LI) was determined by radioimmunoassay in neocortical tissue resected from 20 patients with pharmacologically intractable complex partial seizures. Most resections included either the anterior temporal pole neocortex (15 cases) or cingulate gyrus neocortex (3 cases). The concentration of SS-LI was lowest in cortical tissue immediately adjacent to cortical tumors. Preoperative electrical recordings suggested that this tissue was the seizure focus. In vitro recordings showed that this tissue also exhibited abnormal hyperexcitable synaptic responses. Higher levels of SS-LI, similar to normal values previously reported in human cortex, were present in non-focal temporal neocortical tissue (resected from patients in whom the seizure focus was in the ipsilateral hippocampus) in which no hyperexcitable synaptic activity was present in vitro. The functional loss of inhibitory transmitters suggested by the low SS-LI levels might provide a theoretical basis for the hyperexcitability observed in vivo and in vitro.

Effects of CNS stimulants on the in vivo release of the colocalized transmitters, dopamine and neurotensin, from rat prefrontal cortex.

The effect of CNS stimulant drugs on the in vivo release of the colocalized neurotransmitters dopamine and neurotensin in rat prefrontal cortex was studied using microdialysis. Amphetamine, methylphenidate and nomifensine all increased extracellular fluid (ECF) levels of dopamine; however, their effects of neurotensin varied. Amphetamine increased both ECF dopamine (514 +/- 82% of basal) and neurotensin (350 +/- 49% of basal); however, the neurotensin increase lagged behind the increase in dopamine suggesting a possible trans-synaptic effect. Methylphenidate increased both dopamine and neurotensin (226 +/- 26% and 151 +/- 14% of basal respectively) co-synchronously, suggesting exocytosis of vesicles containing both dopamine and neurotensin. The nomifensine-induced increase in dopamine (202 +/- 23% of basal) was similar to that of methylphenidate, whereas the increase in neurotensin was significantly delayed and of lower magnitude (134 +/- 20% of basal). These data suggest that dopamine and neurotensin in part share a common releaseable pool in the prefrontal cortex. Moreover, dopamine may act presynaptically to increase neurotensin release and the different behavioral profiles of these psychostimulants may in part relate to their different effects on neurotensin release.