In spite of the recognition that striatal D(2) receptors are critical determinants in a variety of psychomotor disorders, the cellular mechanisms by which these receptors shape neuronal activity have remained a mystery. The studies presented here reveal that D(2) receptor stimulation in enkephalin-expressing medium spiny neurons suppresses transmembrane Ca(2+) currents through L-type Ca(2+) channels, resulting in diminished excitability. This modulation is mediated by G(beta)(gamma) activation of phospholipase C, mobilization of intracellular Ca(2+) stores, and activation of the calcium-dependent phosphatase calcineurin. In addition to providing a unifying mechanism to explain the apparently divergent effects of D(2) receptors in striatal medium spiny neurons, this novel signaling linkage provides a foundation for understanding how this pivotal receptor shapes striatal excitability and gene expression.

The computational power of single neurons is greatly enhanced by active dendritic conductances that have a large influence on their spike activity. In cortical output neurons such as the large pyramidal cells of layer 5 (L5), activation of apical dendritic calcium channels leads to plateau potentials that increase the gain of the input/output function and switch the cell to burst-firing mode. The apical dendrites are innervated by local excitatory and inhibitory inputs as well as thalamic and corticocortical projections, which makes it a formidable task to predict how these inputs influence active dendritic properties in vivo. Here we investigate activity in populations of L5 pyramidal dendrites of the somatosensory cortex in awake and anaesthetized rats following sensory stimulation using a new fibre-optic method for recording dendritic calcium changes. We show that the strength of sensory stimulation is encoded in the combined dendritic calcium response of a local population of L5 pyramidal cells in a graded manner. The slope of the stimulus-response function was under the control of a particular subset of inhibitory neurons activated by synaptic inputs predominantly in L5. Recordings from single apical tuft dendrites in vitro showed that activity in L5 pyramidal neurons disynaptically coupled via interneurons directly blocks the initiation of dendritic calcium spikes in neighbouring pyramidal neurons. The results constitute a functional description of a cortical microcircuit in awake animals that relies on the active properties of L5 pyramidal dendrites and their very high sensitivity to inhibition. The microcircuit is organized so that local populations of apical dendrites can adaptively encode bottom-up sensory stimuli linearly across their full dynamic range.

The apical tuft of layer 5 pyramidal neurons is innervated by a large number of inhibitory inputs with unknown functions. Here, we studied the functional consequences and underlying molecular mechanisms of apical inhibition on dendritic spike activity. Extracellular stimulation of layer 1, during blockade of glutamatergic transmission, inhibited the dendritic Ca2+ spike for up to 400 ms. Activation of metabotropic GABAB receptors was responsible for a gradual and long-lasting inhibitory effect, whereas GABAA receptors mediated a short-lasting (approximately 150 ms) inhibition. Our results suggest that the mechanism underlying the GABAB inhibition of Ca2+ spikes involves direct blockade of dendritic Ca2+ channels. By using knockout mice for the two predominant GABAB1 isoforms, GABAB1a and GABAB1b, we showed that postsynaptic inhibition of Ca2+ spikes is mediated by GABAB1b, whereas presynaptic inhibition of GABA release is mediated by GABAB1a. We conclude that the molecular subtypes of GABAB receptors play strategically different physiological roles in neocortical neurons.

GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-β A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.

Chadman, K. K. et al. Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice.

Calcium influx into the dendritic tufts of layer 5 neocortical pyramidal neurons modifies a number of important cellular mechanisms. It can trigger local synaptic plasticity and switch the firing properties from regular to burst firing. Due to methodological limitations, our knowledge about Ca2+ spikes in the dendritic tuft stems mostly from in vitro experiments. However, it has been speculated that regenerative Ca2+ events in the distal dendrites correlate with distinct behavioral states. Therefore it would be most desirable to be able to record these Ca2+ events in vivo, preferably in the behaving animal. Here, we present a novel approach for recording Ca2+ signals in the dendrites of populations of layer 5 pyramidal neurons in vivo, which ensures that all recorded fluorescence changes are due to intracellular Ca2+ signals in the apical dendrites. The method has two main features: 1) bolus loading of layer 5 with a membrane-permeant Ca2+ dye resulting in specific loading of pyramidal cell dendrites in the upper layers and 2) a fiberoptic cable attached to a gradient index lens and a prism reflecting light horizontally at 90 degrees to the angle of the apical dendrites. We demonstrate that the in vivo signal-to-noise ratio recorded with this relatively inexpensive and easy-to-implement fiberoptic-based device is comparable to conventional camera-based imaging systems used in vitro. In addition, the device is flexible and lightweight and can be used for recording Ca2+ signals in the distal dendritic tuft of freely behaving animals.

Key points• Voltage-dependent Ca 2+ channels mediate a large repertoire of physiological actions, including the generation of dendritic spikes in neocortical pyramidal neurons; however, the type of Ca 2+ channels involved in their generation remains unknown.• We found that L-type Ca 2+ currents generate the sustained plateau potential of the Ca 2+ spike. GABA B receptors inhibit Ca 2+ spikes by specifically blocking dendritic L-type currents.• This inhibition is mediated by a direct G i/o -βγ-subunit interaction with the Ca v 1 channels.• Protein kinases (protein kinase C and A) have an important influence on the generation and sustaining of dendritic Ca 2+ spikes; however, their activity is not involved in the GABA B -mediated inhibition of Ca 2+ spikes.• Inhibitory modulation of dendritic activity is important to understand the transformation of synaptic inputs into neuronal output activity. Our results shed light on the molecular mechanisms by which GABA acting via its GABA B receptors can exert this inhibitory action.Abstract Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABA B receptors inhibit calcium-mediated electrogenesis (Ca 2+ spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca 2+ spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca 2+ imaging we found that all subtypes of VDCCs were present in the Ca 2+ spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca 2+ spikes. Particularly, Ca v 1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca 2+ spike. Activation of GABA B receptors specifically inhibited Ca v 1 channels. This inhibition of L-type Ca 2+ currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the G i/o -βγ-subunit with Ca v 1 channels identifying this mechanism as the general pathway of GABA B receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the Abbreviations 4-AP, 4-aminopyridine; ACSF, artificial cerebral spinal fluid; AP, action potential; G i/o -βγ, βγ-subunit of a G i/o protein; PI3K, phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinases I and II; PKC, protein kinase C; PLC, phospholipase C; TEA, tetraethylammonium-chloride; VDCCs, voltage-dependent calcium channels.

. The modulatory effect of D 2 dopamine receptor activation on calcium currents was studied in neostriatal projection neurons at two stages of rat development: postnatal day (PD)14 and PD40. D 2 -class receptor agonists reduced whole cell calcium currents by about 35% at both stages, and this effect was blocked by the D 2 receptor antagonist sulpiride. Nitrendipine partially occluded this modulation at both stages, indicating that modulation of Ca V 1 channels was present throughout this developmental interval. Nevertheless, modulation of Ca V 1 channels was significantly larger in PD40 neurons. -Conotoxin GVIA occluded most of the Ca 2ϩ current modulation in PD14 neurons. However, this occlusion was greatly decreased in PD40 neurons. -Agatoxin TK occluded a great part of the modulation in PD40 neurons but had a negligible effect in PD14 neurons. The data indicate that dopaminergic D 2 -mediated modulation undergoes a change in target during development: from Ca V 2.2 to Ca V 2.1 Ca 2ϩ channels. This change occurred while Ca V 2.2 channels were being down-regulated and Ca V 2.1 channels were being up-regulated. Presynaptic modulation mediated by D 2 receptors reflected these changes; Ca V 2.2 type channels were used for release in young animals but very little in mature animals, suggesting that changes took place simultaneously at the somatodendritic and the synaptic membranes.