Glutamatergic signaling and intracellular calcium mobilization in the spinal cord are crucial for the development of nociceptive plasticity, which is associated with chronic pathological pain. Long-form Homer proteins anchor glutamatergic receptors to sources of calcium influx and release at synapses, which is antagonized by the short, activity-dependent splice variant Homer1a. We show here that Homer1a operates in a negative feedback loop to regulate the excitability of the pain pathway in an activity-dependent manner. Homer1a is rapidly and selectively upregulated in spinal cord neurons after peripheral inflammation in an NMDA receptor-dependent manner. Homer1a strongly attenuates calcium mobilization as well as MAP kinase activation induced by glutamate receptors and reduces synaptic contacts on spinal cord neurons that process pain inputs. Preventing activity-induced upregulation of Homer1a using shRNAs in mice in vivo exacerbates inflammatory pain. Thus, activity-dependent uncoupling of glutamate receptors from intracellular signaling mediators is a novel, endogenous physiological mechanism for counteracting sensitization at the first, crucial synapse in the pain pathway. Furthermore, we observed that targeted gene transfer of Homer1a to specific spinal segments in vivo reduces inflammatory hyperalgesia. Thus, Homer1 function is crucially involved in pain plasticity and constitutes a promising therapeutic target for the treatment of chronic inflammatory pain.

Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS.

Intracellular calcium mobilization and signaling mechanisms triggered by activation of synaptic glutamate receptors in the striatum are important modulators of neurotransmission in striatal circuits. However, the expression and functions of scaffolding proteins anchoring glutamate receptors at striatal synapses have not been addressed so far. The long-form Homer1 proteins, Homer1b͞c, assemble group I metabotropic glutamate receptors (mGluR1͞5) in large macromolecular complexes with sources of calcium influx and release at synapses as well as with components of the NMDA receptor complex at the neuronal cell membrane. Homer1a, the short, activitydependent splice variant of Homer1b͞c, lacks the ability of linking mGluR1͞5 to synaptic proteins and functions as an endogenous negative modulator of the mGluR1͞5 inositol 1,4,5-trisphosphate receptor signaling complex. We have generated transgenic mice, which overexpress Homer1a in striatal medium spiny neurons either homogenously throughout the extrastriosomal matrix (Homer1a-matrix line) or predominantly in striosomal patches (Homer1a-striosome line). Homer1a-expressing mice demonstrated normal development of striatal structure and afferent-efferent connectivity. However, motor performance in behavioral tasks and striatal responses to the psychomotor stimulant amphetamine were significantly altered in the Homer1a-striosome line. Thus, glutamate receptor scaffolding proteins of the Homer1 family critically regulate the functions of striatal medium spiny neurons in complex motor tasks and its modulation by psychomotor stimulant drugs. metabotropic glutamate ͉ motor stereotypy ͉ striosome G roup I metabotropic glutamate receptors (mGluR1͞5) are linked to the activation of phospholipase C and generally mediate excitatory effects by eliciting a release of calcium from intracellular stores. mGluR1͞5 are important positive modulators of synaptic function and neuronal activity and have been linked to long-term changes in synaptic function such as long-term potentiation in the hippocampal circuitry (1), long-term depression in the cerebellum (2), drug-induced plasticity in the nucleus acumbens (3), etc. In the striatum, mGluR1͞5 are primarily expressed postsynaptically in striatal projection neurons and subpopulations of interneurons but can also mediate presynaptic effects on thalamostriatal and corticostriatal glutamatergic afferents as well as nigrostriatal dopaminergic afferents (4). The localization and the functions of mGluR1͞5 are strongly dependent on protein-protein interactions with the Homer (Vesl) family of synaptic scaffolding proteins (5-8). In the nervous system, Homer1 proteins function as molecular bridges linking both mGluR1 and mGluR5 to the inositol 1,4,5-trisphosphate receptors (IP 3 Rs) on the endoplasmic reticulum (7-9) and to additional synaptic ion channels such as calcium channels (7) and transient receptor potential channels (10) as well as to components of the NMDA receptor signaling complex by virtue of their ability to form multimers (11-13). An ad...

Small GTPases of the Rho-family, like Rho, Rac and Cdc42, are involved in neuronal morphogenesis by regulating growth cone morphology or dendritic spine formation. G-proteins of the G12-family, G12 and G13, couple G-protein-coupled receptors (GPCRs) to the activation of RhoA. Recently, two novel Rho-specific guanine nucleotide exchange factors (RhoGEFs), PDZ-RhoGEF and LARG, have been identified to interact with the activated alpha-subunits of G12/G13 and are thus believed to mediate GPCR-induced Rho activation. Although studies in neuronal cell lines have shown that G12/G13 and PDZ-RhoGEF mediate GPCR-induced neurite retraction, the role, as well as the expression of this signalling pathway, in intact brain has not been adequately studied. In the present study, we have characterized systematically the expression of G(alpha)12, G(alpha)13, PDZ-RhoGEF and LARG in various murine tissues as well as their subcellular localization in the central and peripheral nervous systems. By performing immunohistochemistry, using polyclonal antibodies raised against the above proteins, we observed that G(alpha)12, G(alpha)13 and their RhoGEF-effectors are distributed widely in the mammalian nervous system. Moreover, these proteins localize to distinct morphological compartments within neurons. While LARG and G(alpha)12 were mainly found in somata of the neurons, PDZ-RhoGEF and G(alpha)13 were predominantly localized in the neuropil of central neurons. Interestingly, PDZ-RhoGEF is a neural-specific protein, whereas LARG is nearly ubiqoutous. Our data provide evidence that the G12/13-RhoGEF-mediated pathway is present throughout the adult brain and may be involved in regulation of neuronal morphogenesis and function via GPCRs.

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