C. Peter Bengtson

Involvement of transient receptor potential‐like channels in responses to mGluR‐I activation in midbrain dopamine neurons

We investigated the involvement of store‐operated channels (SOCs) and transient receptor potential (TRP) channels in the response to activation of the group I metabotropic glutamate receptor subtype 1 (mGluR1) with the agonist (S)‐3,5‐dihydroxyphenylglycine (DHPG, puff application) in dopamine neurons in rat brain slices. The mGluR1‐induced conductance reversed polarity close to 0 mV and at more positive potentials when extracellular potassium concentrations were increased, indicating the involvement of a cationic channel. DHPG currents but not intracellular calcium responses were reduced by low extracellular sodium concentrations but were not affected by sodium channel blockers, tetrodotoxin and saxitoxin or by inhibition of the h‐current with cesium. Abolition of calcium responses with intracellular BAPTA (1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid; 10 mm) did not affect current responses, indicating they were not calcium activated. Extracellular application of non‐selective SOCs and TRP channel blockers 2‐aminoethoxydiphenylborane (2‐APB), SKF96365, ruthenium red and flufenamic acid (but not gadolinium) reduced DHPG current and calcium responses. Intracellular application of ruthenium red and 2‐APB did not affect DHPG currents, indicating that IP3 and ryanodine receptors did not mediate their actions. Single‐cell PCR revealed the presence of TRPC1 and 5 mRNA in most dopamine neurons and subtypes 3, 4 and 6 in some. Store depletion evoked calcium entry indicative of SOCs, providing the first functional observation of such channels in native central neurons. Store depletion with either cyclopiazonic acid or ryanodine abolished calcium but not current responses to DHPG. The electrophysiological and pharmacological properties of the mGluR1‐induced inward current are consistent with the involvement of TRP channels whereas calcium responses are dependent on the function of SOCs in voltage clamp recordings.

Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity

A simplified cell culture system was developed to study neuronal plasticity. As changes in synaptic strength may alter network activity patterns, we grew hippocampal neurones on a microelectrode array (MEA) and monitored their collective behaviour with 60 electrodes simultaneously. We found that exposure of the network for 15 min to the GABAA receptor antagonist bicuculline induced an increase in synaptic efficacy at excitatory synapses that was associated with an increase in the frequency of miniature AMPA receptor‐mediated EPSCs and a change in network activity from uncoordinated firing of neurones (lacking any recognizable pattern) to a highly organized, periodic and synchronous burst pattern. Induction of recurrent synchronous bursting was dependent on NMDA receptor activation and required extracellular signal‐regulated kinase (ERK)1/2 signalling and translation of pre‐existing mRNAs. Once induced, the burst pattern persisted for several days; its maintenance phase (> 4 h) was dependent on gene transcription taking place in a critical period of 120 min following induction. Thus, cultured hippocampal neurones display a simple, transcription and protein synthesis‐dependent form of plasticity. The non‐invasive nature of MEA recordings provides a significant advantage over traditional assays for synaptic connectivity (i.e. long‐term potentiation in brain slices) and facilitates the search for activity‐regulated genes critical for late‐phase plasticity.

Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis

Various evidence suggests that amyotrophic lateral sclerosis (ALS) selectively affects motor neuron functioning, but electrophysiological alterations of single motor neurons in ALS remains to be documented. In the present work, the excitability of motor neurons has been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly(93)-->Ala). Patch-clamp recordings of membrane potential in transgenic mice motor neurons showed that they fire with increased frequency and shorter duration compared to motor neurons from control mice. The passive membrane properties of these neurons were equivalent however. Such results suggest that an altered motor neuron excitability accompanies an ALS associated mutation and that may contribute to the pathogenesis of the disease.

Synaptic Activity Induces Dramatic Changes in the Geometry of the Cell Nucleus: Interplay between Nuclear Structure, Histone H3 Phosphorylation, and Nuclear Calcium Signaling

Synaptic activity initiates many adaptive responses in neurons. Here we report a novel form of structural plasticity in dissociated hippocampal cultures and slice preparations. Using a recently developed algorithm for three-dimensional image reconstruction and quantitative measurements of cell organelles, we found that many nuclei from hippocampal neurons are highly infolded and form unequally sized nuclear compartments. Nuclear infoldings are dynamic structures, which can radically transform the geometry of the nucleus in response to neuronal activity. Action potential bursting causing synaptic NMDA receptor activation dramatically increases the number of infolded nuclei via a process that requires the ERK-MAP kinase pathway and new protein synthesis. In contrast, death-signaling pathways triggered by extrasynaptic NMDA receptors cause a rapid loss of nuclear infoldings. Compared with near-spherical nuclei, infolded nuclei have a larger surface and increased nuclear pore complex immunoreactivity. Nuclear calcium signals evoked by cytosolic calcium transients are larger in small nuclear compartments than in the large compartments of the same nucleus; moreover, small compartments are more efficient in temporally resolving calcium signals induced by trains of action potentials in the theta frequency range (5 Hz). Synaptic activity-induced phosphorylation of histone H3 on serine 10 was more robust in neurons with infolded nuclei compared with neurons with near-spherical nuclei, suggesting a functional link between nuclear geometry and transcriptional regulation. The translation of synaptic activity-induced signaling events into changes in nuclear geometry facilitates the relay of calcium signals to the nucleus, may lead to the formation of nuclear signaling microdomains, and could enhance signal-regulated transcription.

Transient receptor potential‐like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones

Transient receptor potential (TRP) channels form cationic channels activated by diverse factors including mechanical stimuli, changes in osmolarity, pH and temperature, as well as the exogenous irritant, capsaicin. Metabotropic glutamate receptors have also recently been linked to TRP channel activation in neurones of the substantia nigra, hippocampus and cerebellum, suggesting a novel role for such channels in synaptic communication via endogenous neurotransmitters. We tested this for dopamine neurones in rat brain slices by characterizing the current–voltage relationship and pharmacology of EPSCs mediated by group I metabotropic glutamate receptor subtype 1 (mGluR1). Slow inward currents (273 ± 35 pA peak amplitude, 381 ± 25 ms latency, holding potential (Vh) =−73 mV) representing evoked mGluR1 EPSCs were isolated in the presence of antagonists of AMPA, NMDA, GABAA, GABAB, muscarinic and glycine receptors. CPCCOEt (100 μm), an mGluR1 antagonist, blocked the residual EPSC in all recordings. mGluR1‐activated EPSCs reversed polarity near −10 mV, consistent with the involvement of a cationic channel. Extracellular application of the non‐selective TRP channel blockers SKF 96365, flufenamic acid and ruthenium red caused reversible inhibition of mGluR1‐activated EPSCs. These characteristics parallel those of mGluR1 activation with an agonist and indicate the involvement of a TRP‐like channel in mGluR1‐mediated EPSCs.

Nuclear Calcium Signaling

Calcium is the major intracellular messenger linking synaptic activity in neurons to gene expression to control diverse functions including adaptive responses to synaptic activity as well as survival and death (Bading et al. 1993; Hardingham et al. 1997; Chawla and Bading 2001; West et al. 2001; Zhang et al. 2007; Flavell and Greenberg 2008; Mellstrom et al. 2008; Redmond 2008; Wayman et al. 2008; Bootman et al. 2009; Zhang et al. 2009; Hardingham and Bading 2010). Calcium entry at the synapse acts locally to activate signaling cascades which regulate posttranslational modifications essential for synaptic plasticity, such as the insertion of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic membrane (Soderling 2000; Malinow and Malenka 2002; Ehrlich and Malinow 2004). Synaptic activity can also evoke calcium signals in the nucleus which regulate gene pools largely through the phosphorylation of cAMP response element-binding protein (CREB) and its coactivator, CREB-binding protein (CBP) (Bading et al. 1993; Hardingham et al. 1997; Hardingham et al. 1999; Hu et al. 1999; Hardingham et al. 2001b; Impey et al. 2002; Zhang et al. 2009). Distinct mechanisms have been proposed to mediate synaptically generated calcium signals in subcompartments of pyramidal neurons; N-methyl-D -aspartate receptors (NMDARs) and ryanodine receptors have been implicated in the spine, inositol 3,4,5 triphosphate (IP3) receptors in the dendrites, and L-type voltage-gated calcium channels (VGCCs) at the soma and nucleus, although both NMDARs and IP3 receptors can also contribute to somatic and nuclear calcium signals under certain stimulation conditions (Nakamura et al. 1999; Bardo et al. 2006; Raymond and Redman 2006; Watanabe et al. 2006; Hong and Ross 2007; Hagenston et al. 2008; Bengtson et al. 2010). We review here the calcium signaling pathways underlying synaptically activated gene transcription leading to long-lasting changes in synaptic efficacy and memory as well as the physiological mechanisms by which synaptic activity evokes nuclear calcium signals.

Down-regulation of nitrergic transmission in the rat striatum after chronic nigrostriatal deafferentation.

Dopamine and NO are physiological stimulators of synthesis of cAMP and cGMP, respectively, and NO synthase‐containing interneurons in the striatum are physiologically activated by dopamine‐containing neurons in the substantia nigra. This study investigated whether lesioning dopamine neurons has multiple consequences in the striatum consistent with the reported sensitization of cAMP synthesis, including alteration of the NO–cGMP pathway and phosphodiesterase‐dependent metabolism of cyclic nucleotides. The substantia nigra of adult Sprague‐Dawley rats was unilaterally lesioned with 6‐hydroxydopamine. Two months later, we determined expression of NO synthase and evaluated cGMP and cAMP levels of intact and deafferented striatum. Moreover, we evaluated cAMP– and cGMP–phosphodiesterase activities in basal conditions and after Ca2+–calmodulin stimulation and determined the expression of the phosphodiesterase‐1B isoform and the levels of phosphodiesterase‐1B mRNA. Using immunocytochemistry we characterized the distribution of NO synthase and phosphodiesterase‐1B within striatal neurons. In the dopamine‐deafferented striatum, NO synthase levels were decreased by 42% while NO synthase‐immunopositive intrastriatal fibres but not NO synthase neuronal bodies were reduced in number. In the deafferented striatum basal cGMP levels were reduced, and cAMP levels were increased, but cGMP–phosphodiesterase and cAMP–phosphodiesterase activities were both increased in basal and Ca2+–calmodulin‐stimulated conditions. Accordingly, phosphodiesterase‐1B expression and phosphodiesterase‐1B mRNA were upregulated while a large population of medium‐sized striatal neurons showed increased phosphodiesterase‐1B immunoreactivity. Dopamine deafferentation led to a complex down‐regulation of the NO–cGMP pathway in the striatum and to an up‐regulation of phosphodiesterase‐1B‐dependent cyclic nucleotide metabolism, showing new aspects of neuronal plasticity in experimental hemiparkinsonism.

Nuclear Calcium-VEGFD Signaling Controls Maintenance of Dendrite Arborization Necessary for Memory Formation

The role of neuronal dendrites is to receive and process synaptic inputs. The geometry of the dendritic arbor can undergo neuronal activity-dependent changes that may impact the cognitive abilities of the organism. Here we show that vascular endothelial growth factor D (VEGFD), commonly known as an angiogenic mitogen, controls the total length and complexity of dendrites both in cultured hippocampal neurons and in the adult mouse hippocampus. VEGFD expression is dependent upon basal neuronal activity and requires nuclear calcium-calmodulin-dependent protein kinase IV (CaMKIV) signaling. Suppression of VEGFD expression in the mouse hippocampus by RNA interference causes memory impairments. Thus, nuclear calcium-VEGFD signaling mediates the effect of neuronal activity on the maintenance of dendritic arbors in the adult hippocampus and is required for cognitive functioning. These results suggest that caution be employed in the clinical use of blockers of VEGFD signaling for antiangiogenic cancer therapy.

Diffusion and not active transport underlies and limits ERK1/2 synapse-to-nucleus signaling in hippocampal neurons

The propagation of signals from synapses and dendrites to the nucleus is crucial for long lasting adaptive changes in the nervous system. The ERK-MAPK pathway can link neuronal activity and cell surface receptor activation to the regulation of gene transcription, and it is often considered the principal mediator of synapse-to-nucleus communication in late-phase plasticity and learning. However, the mechanisms underlying ERK1/2 trafficking in dendrites and nuclear translocation in neurons remain to be determined leaving it unclear whether ERK1/2 activated at the synapse can contribute to nuclear signaling and transcriptional regulation. Using the photobleachable and photoactivable fluorescent tag Dronpa on ERK1 and ERK2, we show here that ERK1/2 translocation to the nucleus of hippocampal neurons is induced by the stimulation of N-methyl-d-aspartate receptors or TrkB stimulation and is apparently mediated by facilitated diffusion. In contrast, ERK1/2 trafficking within dendrites is not signal-regulated and is mediated by passive diffusion. Within dendrites, the reach of a locally activated pool of ERK1/2 is very limited and follows an exponential decay with distance. These results indicate that successful signal propagation to the nucleus by the ERK-MAPK pathway depends on the distance of the nucleus from the site of ERK1/2 activation. ERK1/2 activated within or near the soma may rapidly reach the nucleus to induce gene expression, whereas ERK1/2 activated at distal synapses may only contribute to local signaling.

Analysis of stem cell lineage progression in the neonatal subventricular zone identifies EGFR+/NG2- cells as transit-amplifying precursors.

In the adult subventricular zone (SVZ), astroglial stem cells generate transit‐amplifying precursors (TAPs). Both stem cells and TAPs form clones in response to epidermal growth factor (EGF). However, in vivo, in the absence of sustained EGF receptor (EGFR) activation, TAPs divide a few times before differentiating into neuroblasts. The lack of suitable markers has hampered the analysis of stem cell lineage progression and associated functional changes in the neonatal germinal epithelium. Here we purified neuroblasts and clone‐forming precursors from the neonatal SVZ using expression levels of EGFR and polysialylated neural cell adhesion molecule (PSANCAM). As in the adult SVZ, most neonatal clone‐forming precursors did not express the neuroglia proteoglycan 2 (NG2) but displayed characteristics of TAPs, and only a subset exhibited antigenic characteristics of astroglial stem cells. Both precursors and neuroblasts were PSANCAM+; however, neuroblasts also expressed doublecortin and functional voltage‐dependent Ca2+ channels. Neuroblasts and precursors had distinct outwardly rectifying K+ current densities and passive membrane properties, particularly in precursors contacting each other, because of the contribution of gap junction coupling. Confirming the hypothesis that most are TAPs, cell tracing in brain slices revealed that within 2 days the majority of EGFR+ cells had exited the cell cycle and differentiated into a progenitor displaying intermediate antigenic and functional properties between TAPs and neuroblasts. Thus, distinct functional and antigenic properties mark stem cell lineage progression in the neonatal SVZ. STEM CELLS 2009;27:1443–1454