A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.

The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.

Synaptic clustering of Fascilin II and Shaker: essential targeting sequences and role of Dlg.

Previous studies have shown that both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Here, we use the GAL4-UAS system to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker and show that the C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. Moreover, we show that the PDZ-containing protein Discs-Large (Dlg) controls the localization of these proteins, most likely through a direct interaction with their C-terminal amino acids. Finally, transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane.

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.

Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

The dopamine innervation of the visceral cortex mediates the aversive effects of opiates.

We have previously reported that opiates acting on peripheral receptors produce aversive effects whereas opiates acting on central brain receptors produce rewarding effects. The neurotransmitter dopamine (DA) has previously been implicated in both opiate reinforcing (positive) and aversive (negative) effects. We, therefore, chose to investigate the effects of disruption of DA systems on these two motivational properties of the opiate, morphine. Moreover, we sought to determine the brain site where dopamine might act as a mediator of these motivational effects. One group of rats received 6-hydroxydopamine (6-OHDA) lesions of the visceral (agranular insular) cortex to destroy dopaminergic innervation to this area. A separate group of animals were pretreated with intraperitoneal (IP) injections of the DA receptor blocker, alpha-flupenthixol (0.8 mg/kg), followed in both groups by 15 mg/kg (IP) morphine. Both 6-OHDA-lesioned and alpha-flupenthixol-pretreated subjects failed to develop the normal aversion to saccharin seen in control groups following conditioned taste aversion training with morphine. In a place conditioning paradigm, the aversive effects produced by low IP injections of morphine (acting on peripheral receptors) were blocked by 6-OHDA lesions of the visceral cortex. However, DA depletion of the visceral cortex did not disrupt the ability of animals to acquire a morphine place preference. Taken together, these results indicate that DA innervation of the visceral cortex mediates the aversive, but not the rewarding, properties of opiates.

Peripheral receptors mediate the aversive conditioning effects of morphine in the rat.

Previous evidence has shown that morphine produces positive reinforcing effects (as measured in the place conditioning paradigm) through an action in the central nervous system (CNS). The aversive conditioning effects of morphine (as measured in the place and taste conditioning paradigms) were produced when drug action was restricted to peripheral sites, particularly in the gut region. We now demonstrate that most of the aversive conditioning effects of morphine (using place and taste conditioning paradigms) are receptor mediated effects exerted through an action on peripheral opiate receptors. The conditioned taste aversions induced by intraperitoneal (IP) morphine (15 mg/kg) but not amphetamine (1 mg/kg) were attenuated by low IP doses of opiate antagonists (0.1 mg/kg of naltrexone or 1 mg/kg of the peripherally acting antagonist methynaltrexone (MN]. Morphine-, but not amphetamine-induced conditioned taste aversions were also attenuated in animals whose small sensory neurons, bearing the majority of primary afferent opiate receptors, were destroyed by neonatal treatments with capsaicin. In the place conditioning paradigm, the aversive conditioning effects produced by low IP administrations of morphine were blocked by opiate antagonists. Intraperitoneal pretreatments with 1 mg/kg of the quaternary opiate antagonist MN (which does not cross the blood-brain barrier effectively) were shown to block the conditioned place aversions produced by low IP doses of morphine (0.05 mg/kg), but not the place aversions produced by lithium chloride (75 mg/kg IP), or by high doses of naloxone (10 mg/kg SC). These results demonstrate that the aversive conditioning effects of morphine are primarily mediated through an action on peripheral opiate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of cocaine and heroin self-administration following kainic acid lesions of the nucleus accumbens.

In previous experiments we have demonstrated that bilateral infusions of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens result in a drastic reduction in the rate of cocaine self-administration. If this effect is due to the destruction of a presynaptic dopaminergic element in this nucleus, then selective removal of the postsynaptic neuron should also disrupt cocaine self-administration. This hypothesis was tested using the neurotoxin kainic acid. Bilateral kainic acid infusions into the nucleus accumbens resulted in a drastic destruction of cell bodies yet did not damage catecholamine innervation in areas anterior to the accumbens. The effects of these kainic acid infusions were evaluated in rats that had previously acquired cocaine self-administration behavior. These lesions were found to severely disrupt cocaine intake and the degree of damage produced in the accumbens was found to correlate (r = 0.88) with postlesion cocaine intake. These lesions were additionally found to disrupt apomorphine and heroin self-administration. The possibility that these results are due to destruction of systems necessary for stimulant and opiate reward is discussed.

Evidence on the retrograde neurotoxicity of doxorubicin.

Doxorubicin, a fluorescent retrograde neurotoxin, killed neurons in the ventral tegmentum and thalamus that were afferent to the injection site in the caudate-putamen. The neurotoxic effects in the ventral tegmentum and thalamus were prevented by a large coronal knife cut caudal to the injection site in the striatum, suggesting that retrograde transport of doxorubicin is necessary for the death of afferent neurons. In the striatum the neurotoxic effects of doxorubicin 14 days post-injection were quantitatively much greater on afferent neurons (as assessed by a 72% drop in dopamine levels) than on local striatal perikarya (as evidenced by the small 22% drop in the activity of glutamic acid decarboxylase). Doxorubicin may be a useful tool for selectively destroying, by way of retrograde transport, neurons that are afferent to a site in the brain.

Experimentally induced glucose intolerance increases oral ethanol intake in rats.

A number of studies have shown a relationship between glucose tolerance and ethanol intake. The present study uses a relatively simple procedure to induce glucose intolerance to test whether this condition is sufficient to produce an increase in chronic ethanol intake in male rats. Subjects were divided equally into four groups where they were given access to one of four solutions: peppermint-flavored sucrose (40%), peppermint-flavored saccharin (0.1%), peppermint in water (0.1%), or water alone presented three times a week, for a period of 11 weeks. After 12 weeks all animals were subjected to an oral glucose tolerance test which revealed that the chronically prepared sucrose animals had become glucose intolerant. At the start of week 13 all animals were given access to a 6% ethanol solution flavored with peppermint in place of the previous solutions for a period of 11 weeks. Sucrose animals displayed an immediate preference for ethanol and consumed approximately three times more ethanol than the remaining groups. The results of this study indicate that rats that are made glucose intolerant by long term access to a high concentration of sucrose, when given the opportunity, will subsequently choose to drink more ethanol than control animals.

Establishment of secondary reinforcement in sign tracking and place preference tests following pimozide treatment.

The effects of pimozide (1.0 mg/kg), a DA receptor blocker, on the capacity of environmental stimuli to acquire secondary reinforcing properties was investigated using two different paradigms. In the first experiment rats pretreated with either pimozide or its vehicle, were exposed to light-food pairings. When tested under drug-free extinction conditions, these animals approached the light cue significantly more frequently than did control animals who never had the cue associated with food during training. No differences in approach behavior were observed between the pimozide and vehicle groups that received the light-food pairings. The second experiment employed a place preference paradigm where animals were confined in distinctive compartments under reinforced (S+) or nonreinforced (S-) conditions. Pimozide and vehicle treated animals, when tested drug-free and given unrestricted access to both chambers under extinction conditions, spent comparable amounts of time in the S+ chamber relative to vehicle subjects that had never received food in either chamber. The results from these two studies indicate that an animal's ability to code relevant environmental information and to use this encoded information to guide and direct food seeking behavior is relatively independent of dopaminergic activity. The results also have significance for any theory which assumes that dopamine mediates reward processes.