Claudia Wiedemann

Neuronal Networks: A hub of activity

Vesicle recycling has an important role in maintaining circadian clock gene expression in the SCN.activation of neuronal assemblies is essential for the establishment of proper network wiring. Theoretically, a few highly connected neurons with long-ranging connectivity, called ‘hub neurons’, would be the most efficient way to orchestrate network-wide synchronicity, but the existence of such cells had not been proven. Now, Cossart and colleagues have shown that subpopulations of GABA (γ-aminobutyric acid)ergic interneurons act as hub neurons in hippocampal slices. Giant depolarizing potentials, which result from synchronization of activity across the hippocampal network, originate in CA3, making the hippocampus an ideal system for investigating the existence of hub neurons. The authors used a calcium indicator to study calcium dynamics in CA3 in hippocampal slice preparations from 5–7-day-old rats. They constructed a functional connectivity map of all recoded neurons by considering a connection between neurons A and B as functional and directional if activation of A always preceded activation of B. Only a few neurons were functionally connected to a high number of other cells, suggesting that these might be hub neurons. To test how neurons with different degrees of connectivity contribute to the network dynamics, the authors stimulated neurons and simultaneously imaged population activity in real time. Some of the cells with a high degree of connectivity (8 out of 20 cells) stimulated network interactions, suggesting that they function as hub neurons. The hippocampus comprises mainly two cell types, namely glutamatergic pyramidal cells and GABAergic interneurons. Investigation of hippocampal slices from transgenic mice in which hippocampal interneurons were fluorescently labelled revealed that all neurons fulfilling the criteria of hub neurons belonged to this neuronal population. Further analysis found that these hub neurons had one of two specific axonal morphologies: long-reaching connections and sparse collaterals, or a dense and local arborization. Only cells with the dense and local arborization could trigger network synchronization; however, it is possible that long-reaching connections had been cut off during slice preparation. This paper reveals evidence that GABAergic interneurons function as hub neurons and narrows the gap in our understanding of communication between single cells and synchronization of network activity. Claudia Wiedemann

Development: Scaling with microRNAs

erage of their receptive fields during development, growth of the dendritic arbour in synchrony with body growth (dendritic scaling) is essential to maintain the coverage. How this proportional growth of dendrites is coordinated is largely unknown. A study by Jan and colleagues now shows that expression of the microRNA gene bantam in epithelial cells regulates dendritic scaling in peripheral Drosophila melanogaster class IV dendritic arbour (da) neurons through downregulation of Akt signalling. Focusing on class IV da neurons, because of their elaborate arbours, the authors screened D. melanogaster mutants for defects in dendritic growth, and identified the most severe effects in flies lacking bantam. Class IV da neurons in these mutants exhibited disproportional dendritic growth during scaling. Not only were the dendrites longer than normal in these cells, they were greater in number and density as well. The authors then monitored dendrite growth throughout larval development. The da neurons of bantam mutants showed normal dendritic growth during the early, rapid growth phase, when receptive fields are established; however, shortly afterwards their dendritic arbours grew larger than those of wild-type da neurons. This suggests that bantam regulates signals that normally restrict dendrite growth. bantam expression was detectable in wild-type larval muscle and epithelial cells as well as in peripheral neurons after receptive fields had been established. Surprisingly, knocking down bantam in single class IV da neurons had no effect on scaling. Moreover, neuronal expression of bantam in the bantamnull flies did not rescue the scaling defect. However, when bantam was expressed in bantam-null epithelial cells the scaling defect was reduced, and the extent of the rescue was dependent on the level of bantam expression. In addition, overexpression of bantam in wild-type epithelial cells severely reduced dendrite growth. These results are consistent with bantam in epithelial cells regulating dendrite growth-inhibitory signals. Which growth signals are regulated by bantam? Using expression profiling, the authors identified Akt — a well-established regulator of growth — among the numerous candidate genes that were deregulated in neurons of bantamdeficient flies, as Akt expression was increased in these neurons but was normal when bantam was expressed in epithelial cells. Consistent with these results, overexpression or activation of Akt in neurons caused scaling defects similar to those exhibited by bantam mutants. Conversely, knockdown or inhibition of Akt reduced dendritic growth. This indicates that bantam regulates Akt levels in a non-cellautonomous manner in da neurons. The authors also showed that bantam regulates the growth of Class III, but not Class I, da neurons. They suggest that the different time courses of dendritic scaling of these cells might mean that they require different scaling signals. It remains to be seen whether noncell-autonomous regulation through microRNAs is a general mechanism for coordinated dendritic scaling.

Synaptic plasticity: ARC de LTD

(mGluR)-dependent long-term depression (mGluR-LTD) relies on dendritic protein synthesis that occurs within minutes of mGluR activation, but the identities of the synthesized proteins are largely unknown. Two new studies show that the rapid translation of activityregulated cytoskeleton-associated protein (ARC; also termed activityregulated gene of 3.1 kb (ARG3.1)) is essential for mGluR-LTD. Both groups of researchers used hippocampal neuronal cultures and acute slices to investigate the molecular mechanism of mGluR-LTD. Both studies initially established that induction of LTD with the mGluR agonist dihydroxyphenylglycine (DHPG) in hippocampal cultures led to a long-term decrease in surface AMPARs (a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptors). This could be blocked by inhibiting mRNA translation. As Arc/Arg3.1 mRNA is present in dendrites, and as ARC/ARG3.1 is known to stimulate AMPAR endocytosis, both research groups measured dendritic ARC/ARG3.1 protein levels following the stimulation of neurons with DHPG. Both groups observed a significant increase in dendritic ARC/ARG3.1 protein levels. Blocking protein synthesis prevented this increase, whereas blocking DNA transcription did not, indicating that mGluR-LTD requires de novo protein synthesis. Waung et al. obtained similar results with acute hippocampal slices in which the dendrites had been mechanically severed from the nerve cell body, suggesting that ARC/ARG3.1 is locally synthesized from pre-existing, dendritic mRNA. Furthermore, mGluR-LTD was impaired in slices from Arc/Arg3.1knockout mice (Park, Park et al.) or when Arc/Arg3.1 mRNA translation was acutely prevented by antisense oligonucleotides (Waung et al.). The latter finding suggests that rapid translation of Arc/Arg3.1 mRNA is required for mGluR-LTD. Paradoxically, Park, Park et al. revealed that low doses of the protein-synthesis inhibitor cycloheximide increased the levels of ARC/ ARG3.1 protein. This pointed the authors towards eukaryotic translation elongation factor 2 (EEF2), as EEF2 that has been phosphorylated by EEF2 kinase (EEF2K) inhibits elongation in protein synthesis but has been shown to increase the translation of certain mRNAs. Co-immunoprecipitation studies showed that mGluRs directly associate with EEF2K, and that this interaction is reduced by mGluR activation. Park, Park et al. investigated hippocampal slices from Eef2kknockout mice, and showed that mGluR-LTD is absent in these slices and that de novo ARC/ARG3.1 synthesis is absent in Eefk2-knockout neurons. The authors concluded that the EEF2K–EEF2–ARC/ARG3.1 pathway is important for mGluR-LTD. In a mouse model of Fragile X syndrome, in which the dendritic mRNA-binding protein fragile X mental retardation protein (FMRP) is mutated and in which Arc/Arg3.1 mRNA translation is de-repressed, mGluR-LTD is abnormal. In neuronal cultures from Fmr1-knockout mice, the increase in ARC/ARG3.1 protein levels after DHPG stimulation was absent. Similarly, in slices from Arc/Arg3.1;Fmr1 double-knockout mice, DHPG-evoked LTD was impaired. This again highlights the importance of the EEF2K–EEF2– ARG/ARG3.1 pathway in mGluR-LTD. These results demonstrate that local, rapid translation of Arc/Arg3.1 mRNA is essential for mGluR-LTD but not for NMDAR (N-methyl-daspartate receptor)-dependent LTD, and give insight into the mechanistic differences between these two forms of LTD. mGluR-LTD might function to mediate adaptive behaviours, as rapid protein synthesis in dendrites probably contributes to synapseselective, long-lasting forms of plasticity. Claudia Wiedemann

Addiction: Cannabis against heroin?

drawal is a big societal problem and is thought to be induced by environmental cues. The identification of treatments that attenuate cue-induced relapse is one aim of addiction research. This study showed that the non-psychotropic component of cannabis, cannabidiol (CBD), antagonizes cue-induced drug-seeking behaviour after heroin withdrawal and discovered that it acts by normalizing glutamate and cannabinoid receptor expression levels in neurons that are involved in the drug-seeking behaviour. The authors investigated the effect of CBD on the maintenance, extinction and relapse of heroin seeking in rats. Animals which were steadily self-administering heroin by pressing a lever in a designated chamber that contained a light as an environmental cue did not change their heroin intake behaviour after treatment with CBD. After 14 days of drug abstinence, rats were re-exposed to the self-administration chamber and stimulus light cue or to the light cue alone. The drug-seeking behaviour of rats that had been treated with CBD 24 hours prior to the behavioural test was attenuated. It is noteworthy that during these tests the rats did not have access to heroin after lever pressing, as that would have reinstated their drug dependence. The results suggest that CBD specifically attenuated drugseeking behaviour in response to environmental cues. The striatum is thought to be involved in reward behaviour and habit formation. The authors found that mRNA and protein levels of the cannabinoid receptor CB1R were significantly increased in the mesolimbic system (specifically the nucleus accumbens (NAc) and dorsal striatum) of heroin-addicted rats but not in the dorsolateral division of the striatum (which receives primarily sensorimotor cortical input). CBDtreated addicted rats had levels of CB1R similar to control animals. These findings are consistent with previous studies which showed that inhibition of CB1R blocks cue-induced drug-seeking behaviour. Drug-seeking behaviour has also been linked to dysregulation of glutamate and opioid transmission. The authors found that the expression of AMPA (α-amino-3-hydroxy5-methyl-4-isoxazole propionic acid) GluR1 receptors, which are strongly linked to drug-seeking behaviour, was downregulated in the NAc core and the medial and lateral shell in heroin-addicted rats. 24 hours after CBD administration, GluR1 protein expression was normalized in the NAc core and medial shell. This study reveals unexpected properties of CBD, namely the selective and prolonged inhibition of cue-induced drug seeking. These characteristics of CBD thus contrast with those of the other main constituent of cannabis, the psycho active Δ9-tetrahydrocannabinol (THC), which has been associated with addiction vulnerability. CBD could therefore be exploited for the development of drugs to combat relapse. Claudia Wiedemann

Neuronal activation: The symphony of transcription

Neuronal activity triggers the activation of thousands of enhancers and the synthesis of enhancer RNA.

Neuron–glia interactions: Do they or don't they?

Modification of astrocytic Ca2+ levels does not affect synaptic transmission in hippocampal slice preparations.

Neuron–glia interactions: With a little help from glia

induction of long-term potentiation (LTP) — a form of synaptic plasticity — in vivo remains controversial. Rusakov and colleagues now show that Ca2+-dependent d-serine release from astrocytes regulates NMDAR (N-methyl-d-aspartate receptor)dependent synaptic plasticity in acute hippocampal slice preparations from adult rats, providing evidence for such an involvement. Co-agonists, such as d-serine and glycine, enable NMDAR activation by glutamate by binding to a co-agonist site of the NMDAR. Cultured astrocytes release d-serine in a Ca2+-dependent manner, and d-serine is required for LTP in culture. To test whether this also applies in a more physiological system — a long-standing controversy in the field — the authors patched individual astrocytes in acute hippocampal slices and monitored field excitatory postsynaptic potentials at synapses between Schaffer collaterals (SCs) and CA1 pyramidal cells. This experimental set-up allowed the authors to monitor and control the intra-astrocytic Ca2+ concentration ([Ca]i) and to introduce pharmacological compounds to regulate d-serine synthesis. High-frequency stimulation of the SCs increases astrocyte [Ca]i. Blocking this increase (by clamping [Ca]i) suppressed LTP at nearby synapses, an effect that was rescued by extracellular addition of d-serine. These results suggest that Ca2+dependent release of an NMDAR co-agonist from astrocytes is essential for the induction of LTP. When the authors blocked the synthesis of d-serine in individual astrocytes and depleted their d-serine pool by high-frequency stimulation, subsequent LTP induction was suppressed, providing direct evidence for a role of astrocyte-derived d-serine in LTP induction. Astrocytes can be connected by gap junction channels, raising the question of how far the influence of a single astrocyte and its associated network on synaptic activity extends into the surrounding area. The authors monitored LTP simultaneously in two neighbouring hippocampal areas that contained two astrocytes. Blocking the increase of [Ca]i in one of the astrocytes suppressed LTP in synapses that were in reach of gap junction-connected astrocytes. Synapses in the vicinity of the other astrocyte, in which Ca2+ rises were allowed, were not affected, demonstrating that the synaptic effects of the two astrocytes could be independent. This study elegantly demonstrated that the induction of LTP at hippocampal SC–CA1 synapses requires Ca2+-dependent release of d-serine from astrocytes. Considering the density of neurons and astrocytes in CA1 in addition to gap junctionconnected astrocytes, d-serine released from a single astrocyte might affect synaptic plasticity of hundreds of neurons nearby. Claudia Wiedemann

Neuron|[ndash]|glia interactions: With a little help from glia

induction of long-term potentiation (LTP) — a form of synaptic plasticity — in vivo remains controversial. Rusakov and colleagues now show that Ca2+-dependent d-serine release from astrocytes regulates NMDAR (N-methyl-d-aspartate receptor)dependent synaptic plasticity in acute hippocampal slice preparations from adult rats, providing evidence for such an involvement. Co-agonists, such as d-serine and glycine, enable NMDAR activation by glutamate by binding to a co-agonist site of the NMDAR. Cultured astrocytes release d-serine in a Ca2+-dependent manner, and d-serine is required for LTP in culture. To test whether this also applies in a more physiological system — a long-standing controversy in the field — the authors patched individual astrocytes in acute hippocampal slices and monitored field excitatory postsynaptic potentials at synapses between Schaffer collaterals (SCs) and CA1 pyramidal cells. This experimental set-up allowed the authors to monitor and control the intra-astrocytic Ca2+ concentration ([Ca]i) and to introduce pharmacological compounds to regulate d-serine synthesis. High-frequency stimulation of the SCs increases astrocyte [Ca]i. Blocking this increase (by clamping [Ca]i) suppressed LTP at nearby synapses, an effect that was rescued by extracellular addition of d-serine. These results suggest that Ca2+dependent release of an NMDAR co-agonist from astrocytes is essential for the induction of LTP. When the authors blocked the synthesis of d-serine in individual astrocytes and depleted their d-serine pool by high-frequency stimulation, subsequent LTP induction was suppressed, providing direct evidence for a role of astrocyte-derived d-serine in LTP induction. Astrocytes can be connected by gap junction channels, raising the question of how far the influence of a single astrocyte and its associated network on synaptic activity extends into the surrounding area. The authors monitored LTP simultaneously in two neighbouring hippocampal areas that contained two astrocytes. Blocking the increase of [Ca]i in one of the astrocytes suppressed LTP in synapses that were in reach of gap junction-connected astrocytes. Synapses in the vicinity of the other astrocyte, in which Ca2+ rises were allowed, were not affected, demonstrating that the synaptic effects of the two astrocytes could be independent. This study elegantly demonstrated that the induction of LTP at hippocampal SC–CA1 synapses requires Ca2+-dependent release of d-serine from astrocytes. Considering the density of neurons and astrocytes in CA1 in addition to gap junctionconnected astrocytes, d-serine released from a single astrocyte might affect synaptic plasticity of hundreds of neurons nearby. Claudia Wiedemann

CSI in SCI: Repair

to promote recovery of motor function after spinal cord injury (SCI) is often very limited. Three studies using rodents now describe several potential new treatment strategies for SCI. The role of infiltrating macrophages and resident microglia in the recovery from SCI has been controversial. Schwartz, Jung and collaborators developed a new method that allowed them to specifically assess the function of monocyte-derived r e pa i r

Neuron–glia interactions: An intimate relationship

and have a vital role in synaptic transmission. This is partly due to the cell surface expression of astroglial glutamate transporters (GLT1; also known as SLC1A2 and EAAT2) that mediate the uptake of extracellular glutamate and thereby modify the intensity of neurotransmission as well as prevent glutamate neurotoxicity. Although it is known that astroglia can regulate neuronal events, such as dendritic spine formation, little is known about the communication from neurons to astroglia. Previous studies have suggested that neuronal activity might regulate astroglial GLT1 expression, but the underlying mechanism was unknown. Publishing in Neuron, Rothstein and colleagues have now revealed that the expression of the transcription factor kappa-B motif-binding phosphoprotein (KBBP) in astrocytes is neurondependent and that KBBP regulates astroglial GLT1 expression. The authors used an in vitro coculture system that kept neurons and astrocytes in different chambers and only allowed axons to make direct contact with astrocytes. Monitoring GLT1 expression in co-cultured astrocytes by immunostaining for GLT1 itself or by using a fluorescent reporter that monitored GLT1 promotor activity revealed that the highest level of GLT1 expression or promotor activity was in astrocytes that were in direct contact with axons. Activation of GLT1 expression was inhibited by blocking neurotransmitter release with tetrodotoxin or glutamate receptor antagonists. These results show that astroglial GLT1 expression is dependent on synaptic interaction and that presynaptic activity has a role in transcriptional activation of GLT1. To evaluate the transcriptional mechanism, the authors analysed the 2.5 kb upstream promoter region of GLT1 using deletion studies and sitedirected mutagenesis. They identified 10 base pairs that were essential for GLT1 promotor activation in vitro and in vivo. Furthermore, they identified KBBP as the transcription factor that binds specifically to this region and demonstrated that KBBP expression correlates with GLT1 expression in mouse astrocytes during synaptogenesis (postnatal days 2–21). KBBP expression in cocultured astrocytes was also induced by axonal contact and highly correlated with GLT1 expression. Silencing KBBP expression in vitro by using small interfering RNA or in vivo by using antisense oligonucleotides reduced GLT1 expression in astrocytes, supporting the notion that KBBP recruitment to the GLT1 promotor is required for GLT1 expression. Next, the authors investigated KBBP and GLT1 expression in in vivo mouse models of denervation and neuronal degeneration. Corticospinal tract transection, neurotoxininduced degeneration of spinal motor neurons and chronic degeneration in an amyotrophic lateral sclerosis mouse model all resulted in the loss of KBBP expression in the affected astroglia and concomitant loss of GLT1, indicating that the integrity of presynaptic terminals helps to maintain astrocyte function. This study revealed part of the neuron-dependent transcriptional mechanism that leads to GLT1 expression in astrocytes. Further studies of transcriptional changes in astrocytes following synaptic disruption will lead to a more complete understanding of how astroglia contribute to normal and diseased brain function and to targets for glial therapy being identified.