Fine structural localization of acetylcholinesterase in electroplaque of the electric eel.

The electroplaques composing the electric organ of the eel, Electrophorus electricus, have been utilized for the dual purpose of demonstrating the subcellular sites of acetylcholinesterase activity and as a model for comparison of the several cytochemical methods available. Fresh tissue and tissue fixed by immersion in formalin, hydroxyadipaldehyde, or glutaraldehyde was reacted with the Cu-thiocholine method, the Cu-ferrocyanide thiocholine method, or the thiolacetic acid (TAA) method using Pb, Ag, or Au as capture reagents. Controls were obtained by omission of substrate, or by addition to complete media of varying concentrations of different cholinesterase inhibitors. Reactions were run at 0-5 degrees C at a pH range of 5.0-7.1 for 0.25 to 120 min. Regardless of the capture metal, the localization obtained with TAA as substrate was identical with that observed with acetylthiocholine, the majority of precipitate being deposited on or near the external innervated surface of the plaque and within the tubulovesicular organelles opening onto the innervated surface. Both of the thiocholine methods and the Pb-TAA method showed reaction product in synaptic vesicles of the nerve endings innervating the plaque which was uninhibitable by 10(-4)M physostigmine. All methods also showed some inhibitor-sensitive deposition of reaction product in the mucoid material forming the immediate extracellular environment of the innervated surface.

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation.

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.

The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

An Internet review: the compleat neuroscientist scours the World Wide Web.

The World Wide Web provides a graphical interface that allows users to explore the multiple databases of information that are accessible on the Internet. In the field of neuroscience, several hundred sites contain potentially pertinent information. This article takes a critical look at those sites and offers recommendations to those seeking broad neuroscience resources as well as those desiring sites specialized for the developmental neurosciences.

Norepinephrine-containing neurons: changes in spontaneous discharge patterns during sleeping and waking.

Norepinephrine-containing neurons of the locus coeruleus of the cat were recorded with microelectrodes during unrestrained sleeping and waking. The recorded neurons were subsequently defined by combined fluorescence histochemistry of catecholamines and production of microlesions at recording sites. These pontine units show homogeneous changes in discharge patterns with respect to sleep stages, firing slowly during drowsy periods and slow wave sleep and firing in rapid bursts during paradoxical sleep. These data provide a direct correlation between the activity of defined catecholamine-containing neurons and the spontaneous occurrence of sleep stages.

Cellular and molecular mechanisms of drug dependence.

The molecular and cellular actions of three classes of abused drugs--opiates, psychostimulants, and ethanol--are reviewed in the context of behavioral studies of drug dependence. The immediate effects of drugs are compared to those observed after long-term exposure. A neurobiological basis for drug dependence is proposed from the linkage between the cellular and behavioral effects of these drugs.

Response artifact in the measurement of neuroleptic-induced anhedonia.

Systemic administration of the neuroleptic drug alpha-flupenthixol attenuated lever-pressing behavior in rats responding for rewarding brain stimulation. The magnitude of this attenuation was dose-dependent and resembled the effects of reward reduction and termination. However, when the operant response requirements of the same rats were changed to nose poking, identical drug treatments produced relatively little attenuation in performance. These data do not support the belief that neuroleptics produce a general state of anhedonia. Rather, the apparent suppression of reinforced behaviors depends at least in part on the kinetic requirements of the response.

Electron-microscopic autoradiography of rat hypothalamus after intraventricular h3-norepinephrine.

Tritiated norepinephrine was injected into the lateral ventricles of rats, and its localization in the hypothalamus was determined by light and electron-microscopic autoradiography. Eighty percent of the autoradiographic grains were located over nerve endings and unmyelinated axons. Large, dense synaptic vesicles were present in most of the endings and axons with activity. Grains were rarely seen over myelinated axons, glia, or blood vessels.

Cytochemistry of synapses: selective staining for electron microscopy.

Material at synapses and in some synaptic vesicles becomes selectively stained when glutaraldehyde-fixed rat brain that has not been treated with osmium is stained with phosphotungstic acid. The material stained at synapses is distinct from the adjacent unstained synaptic membranes and has cytochemical properties of protein. The specialized spatial arrangement exhibited by this synaptic material suggests its close involvement in synaptic function.

Cyclic AMP and cyclic GMP may mediate opposite neuronal responses in the rat cerebral cortex.

Electrophysiologically identified pyramidal tract neurons in the rat cerebral cortex were tested with norepinephrine, acetylcholine, adenosine 3',5'-monophosphate (cyclic AMP), and guanosine 3',5'-monophosphate (cyclic GMP) applied by microiontophoresis. The neurons were usually inhibited by norepinephrine and cyclic AMP, but excited by acetylcholine and cyclic GMP. These opposing responses of pyramidal tract neurons to cyclic AMP and cyclic GMP suggests that these two nucleotides could function as reciprocal intracellular second messengers for norepinephrine and acetylcholine, respectively.

Stimulation of human periaqueductal gray for pain relief increases immunoreactive beta-endorphin in ventricular fluid.

Immunoreactive beta-endorphin was measured in the ventricular fluid of six patients with chronic pain. Stimulation of the periaqueductal gray matter in three patients with pain of peripheral origin resulted in significant increases (50 to 300 percent) in the concentration of ventricular immunoreactive beta-endorphin. In three other patients suffering deafferentation dysesthesia, stimulation of the posterior limb of the internal capsule did not alter the concentration of this peptide. These results provide evidence of the release of human immunoreactive beta-endorphin in vivo and suggest that naloxone-reversible pain relief achieved by stimulation of the periaqueductal gray matter may be in part mediated by the activation of beta-endorphin-rich diencephalic areas.

Systemic ethanol: selective enhancement of responses to acetylcholine and somatostatin in hippocampus.

In rat hippocampal pyramidal cells tested in situ by iontophoresis of several neurotransmitters, ethanol significantly enhanced excitatory responses to acetylcholine and inhibitory responses to somatostatin-14 but had no statistically significant effect on excitatory responses to glutamate or inhibitory responses to gamma-aminobutyric acid or, in preliminary tests, to norepinephrine or serotonin. The effects of ethanol on responses to acetylcholine and somatostatin-14 may provide insight into synaptic mechanisms underlying the behavioral consequences of ethanol intoxication.

GABA catabolism: localization of succinic semialdehyde dehydrogenase in brain motor and sensory nuclei.

The localization of brain succinic semialdehyde dehydrogenase, a specific gamma-aminobutyric acid degradative enzyme, could potentially yield valuable information concerning the function of the enzyme. Application of a new histochemical technique for this enzyme has revealed characteristic patterns of neuronal staining that are contsistent within embryologically and functionally similar nuclei of the brainstem of the rat.

Osmiophilic polymer generation: catalysis by transition metal compounds in ultrastructural cytochemistry.

A transition metal compound that is bound in tissues by any appropriate cytochemical reaction may catalyze the generation of an insoluble osmiophilic polymer from organic monomers such as 3,3'-diaminobenzidine. When the polymers are treated with osmium tetroxide, electron-opaque, insoluble osmium blacks (coordination polymers of osmium) are formed at the sites of the particular macromolecule or enzyme permitting its light, and electron, microscopic localization. This approach represents a distinct advantage over earlier cytochemical methods because the shorter incubation time needed here results in less artifactual deposition of metal ions, and less tendency to crystallize the reaction product. In addition, the shorter incubation times permit longer fixation of tissues and hence less artifact due to enzyme diffusion.

Cyclic adenosine monophosphate: possible mediator for norepinephrine effects on cerebellar Purkinje cells.

Microelectrophoretic application of norepinephrine or cyclic adenosine monophosphate reduces the discharge frequency of Purkinje cells in the rat cerebellum. In contrast, other nucleotides accelerate the discharge rate of most units. Parenterally administered theophylline, which inhibits the hydrolysis of cyclic adenosine monophosphate enhances the effects of norepinephrine and cyclic adenosine monophosphate. Therefore, norepinephrine may be able to regulate Purkinje cells functionally by metabolic stimulation of cyclic adenosine monophosphate synthesis.

Prostaglandins E1 and E2 antagonize norepinephrine effects on cerebellar purkinje cells: microelectrophoretic study.

In microelectrophoretic experiments, prostaglandins E(1) and E(2) antagonize the reduction in discharge rate of cerebellar Purkinje cells produced by norepinephrine. Slowing of discharge evoked by 3',5'-adenosine monophosphate or gamma aminobutyric acid is not antagonized. These data provide the first indication that endogenous prostaglandins may physiologically function to modulate central noradrenergic junctions.

Magnesium pemoline: failure to affect in vivo synthesis of brain RNA.

The effect of magnesium pemoline on the synthesis of brain RNA in vivo was studied. No significant effect either on the concentration of RNA or on the uptake of H(3)-uridine into RNA was detected.

Immunofluorescent localization of cyclic AMP in toad urinary bladder: possible intercellular transfer.

By use of an immunofluorescent cytochemical staining technique, adenosine 3',5'-monophosphate (cyclic AMP) has been localized in toad bladder epithelial cells. Within 2 minutes after addition of vasopressin, staining intensity increases in both mitochondria-rich and granular cells. This finding, taken together with the precise anatomical relation between these two epithelial cell types and the observation that after separation of the two cell types vasopressin stimulates cyclic AMP accumulation in only mitochondria-rich cells, suggests that cyclic AMP may be transferred from mitochrondria-rich to granular cells as part of the response of the toad urinary bladder to vasopressin.

Reversal of diabetes insipidus in Brattleboro rats: intrahypothalamic injection of vasopressin mRNA.

Messenger RNAs occur within the axons of magnocellular hypothalamic neurons known to secrete oxytocin and vasopressin. In Brattleboro rats, which have a genetic mutation that renders them incapable of vasopressin expression and secretion and thus causes diabetes insipidus, injection into the hypothalamus of purified mRNAs from normal rat hypothalami or of synthetic copies of the vasopressin mRNA leads to selective uptake, retrograde transport, and expression of vasopressin exclusively in the magnocellular neurons. Temporary reversal of their diabetes insipidus (for up to 5 days) can be observed within hours of the injection. Intra-axonal mRNAs may represent an additional category of chemical signals for neurons.

Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons.

The atypical excitation by opiates and opioid peptides of hippocampal pyramidal cells can be antagonized by iontophoresis of naloxone, the gamma-aminobutyric acid antagonists bicuculline, or magnesium ion. The recurrent inhibition of these cells evoked by transcallosal stimulation of the contralateral hippocampus is blocked by enkephalin but only shortened by acetylcholine. The results suggest that the opioids excite pyramidal neurons indirectly by inhibition of neighboring inhibitory interneurons (probably containing gamma-aminobutyric acid). This mechanism may be pertinent to the electrographic signs of addictive drugs.