Rafael Luján

Target-cell-specific facilitation and depression in neocortical circuits

In neocortical circuits, repetitively active neurons evoke unitary postsynaptic potentials (PSPs) whose peak amplitudes either increase (facilitate) or decrease (depress) progressively. To examine the basis for these different synaptic responses, we made simultaneous recordings from three classes of neurons in cortical layer 2/3. We induced repetitive action potentials in pyramidal cells and recorded the evoked unitary excitatory (E)PSPs in two classes of GABAergic neurons. We observed facilitation of EPSPs in bitufted GABAergic interneurons, many of which expressed somatostatin immunoreactivity. EPSPs recorded from multipolar interneurons, however, showed depression. Some of these neurons were immunopositive for parvalbumin. Unitary inhibitory (I)PSPs evoked by repetitive stimulation of a bitufted neuron also showed a less pronounced but significant difference between the two target neurons. Facilitation and depression involve presynaptic mechanisms, and because a single neuron can express both behaviors simultaneously, we infer that local differences in the molecular structure of presynaptic nerve terminals are induced by retrograde signals from different classes of target neurons. Because bitufted and multipolar neurons both formed reciprocal inhibitory connections with pyramidal cells, the results imply that the balance of activation between two recurrent inhibitory pathways in the neocortex depends on the frequency of action potentials in pyramidal cells.

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1–A2A Receptor Heteromers

The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, we demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, we showed the existence of A1R–A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R–A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R–A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R–A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine.

Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling

Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (γ-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

Glutamate and GABA receptor signalling in the developing brain.

Our understanding of the role played by neurotransmitter receptors in the developing brain has advanced in recent years. The major excitatory and inhibitory neurotransmitters in the brain, glutamate and GABA, activate both ionotropic (ligand-gated ion channels) and metabotropic (G protein-coupled) receptors, and are generally associated with neuronal communication in the mature brain. However, before the emergence of their role in neurotransmission in adulthood, they also act to influence earlier developmental events, some of which occur prior to synapse formation: such as proliferation, migration, differentiation or survival processes during neural development. To fulfill these actions in the constructing of the nervous system, different types of glutamate and GABA receptors need to be expressed both at the right time and at the right place. The identification by molecular cloning of 16 ionotropic glutamate receptor subunits, eight metabotropic glutamate receptor subtypes, 21 ionotropic and two metabotropic GABA receptor subunits, some of which exist in alternatively splice variants, has enriched our appreciation of how molecular diversity leads to functional diversity in the brain. It now appears that many different types of glutamate and GABA receptor subunits have prominent expression in the embryonic and/or postnatal brain, whereas others are mainly present in the adult brain. Although the significance of this differential expression of subunits is not fully understood, it appears that the change in subunit composition is essential for normal development in particular brain regions. This review focuses on emerging information relating to the expression and role of glutamatergic and GABAergic neurotransmitter receptors during prenatal and postnatal development.

The Increased Trafficking of the Calcium Channel Subunit α2δ-1 to Presynaptic Terminals in Neuropathic Pain Is Inhibited by the α2δ Ligand Pregabalin

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit α2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both α2δ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of α2δ-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, α2δ-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of α2δ-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of α2δ-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of α2δ-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of α2δ-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

Differential Compartmentalization and Distinct Functions of GABAB Receptor Variants

GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses.

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Glutamate receptors on dopamine neurons control the persistence of cocaine seeking

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. Here, we generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. We report that in midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior.

METABOTROPIC GLUTAMATE TYPE 5, DOPAMINE D2 AND ADENOSINE A2A RECEPTORS FORM HIGHER-ORDER OLIGOMERS IN LIVING CELLS

G protein‐coupled receptors are known to form homo‐ and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high‐resolution immunoelectron microscopy we could confirm that the three receptors co‐distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co‐immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu5R, D2R and A2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein‐coupled receptor oligomers in general and the mGlu5R/D2R/A2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.

Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels

Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABAB receptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABAB receptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABAB receptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABAB receptors in vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.