Van B. Lu

Neuron type‐specific effects of brain‐derived neurotrophic factor in rat superficial dorsal horn and their relevance to ‘central sensitization’

Chronic constriction injury (CCI) of the rat sciatic nerve increases the excitability of the spinal dorsal horn. This ‘central sensitization’ leads to pain behaviours analogous to human neuropathic pain. We have established that CCI increases excitatory synaptic drive to putative excitatory, ‘delay’ firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory, ‘tonic’ firing neurons. Here, we use a defined‐medium organotypic culture (DMOTC) system to investigate the long‐term actions of brain‐derived neurotrophic factor (BDNF) as a possible instigator of these changes. The age of the cultures and their 5–6 day exposure to BDNF paralleled the protocol used for CCI in vivo. Effects of BDNF (200 ng ml−1) in DMOTC were reminiscent of those seen with CCI in vivo. These included decreased synaptic drive to ‘tonic’ neurons and increased synaptic drive to ‘delay’ neurons with only small effects on their membrane excitability. Actions of BDNF on ‘delay’ neurons were exclusively presynaptic and involved increased mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both pre‐ and postsynaptic actions on ‘tonic’ cells; mEPSC frequency and amplitude were decreased and the decay time constant reduced by 35%. These selective and differential actions of BDNF on excitatory and inhibitory neurons contributed to a global increase in dorsal horn network excitability as assessed by the amplitude of depolarization‐induced increases in intracellular Ca2+. Such changes and their underlying cellular mechanisms are likely to contribute to CCI‐induced ‘central sensitization’ and hence to the onset of neuropathic pain.

β-Hydroxybutyrate Modulates N-Type Calcium Channels in Rat Sympathetic Neurons by Acting as an Agonist for the G-Protein-Coupled Receptor FFA3

Free fatty acids receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43), for which the short-chain fatty acids (SCFAs) acetate and propionate are agonist, have emerged as important G-protein-coupled receptors influenced by diet and gut flora composition. A recent study (Kimura et al., 2011) demonstrated functional expression of FFA3 in the rodent sympathetic nervous system (SNS) providing a potential link between nutritional status and autonomic function. However, little is known of the source of endogenous ligands, signaling pathways, or effectors in sympathetic neurons. In this study, we found that FFA3 and FFA2 are unevenly expressed in the rat SNS with higher transcript levels in prevertebral (e.g., celiac-superior mesenteric and major pelvic) versus paravertebral (e.g., superior cervical and stellate) ganglia. FFA3, whether heterologously or natively expressed, coupled via PTX-sensitive G-proteins to produce voltage-dependent inhibition of N-type Ca2+ channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that β-hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through FFA3.

N-Arachidonyl Glycine Does Not Activate G Protein–Coupled Receptor 18 Signaling via Canonical Pathways

Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analogue of anandamide, is an endogenous ligand of the Gαi/o protein–coupled receptor 18 (GPR18). However, a high-throughput β-arrestin–based screen failed to detect activation of GPR18 by NAGly (Yin et al., 2009; JBC, 18:12328). To address this inconsistency, this study investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. GPR18 was heterologously expressed in rat sympathetic neurons, and the modulation of N-type (Cav2.2) calcium channels was examined. Proper expression and trafficking of receptor were confirmed by the “rim-like” fluorescence of fluorescently tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents, indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors of Gαi/o-coupled receptors, G protein–coupled inwardly rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple to other G proteins tested: Gαs, Gαz, and Gα15. These results suggest NAGly is not an agonist for GPR18 or that GPR18 signaling involves noncanonical pathways not examined in these studies.

Substantia Gelatinosa neurons in defined-medium organotypic slice culture are similar to those in acute slices from young adult rats.

Abstract Peripheral nerve injury promotes an enduring increase in the excitability of the spinal dorsal horn. This change, that likely underlies the development of chronic pain, may be a consequence of prolonged exposure of dorsal horn neurons to mediators such as neurotrophins, cytokines, and neurotransmitters. The long‐term effects of such mediators can be analyzed by applying them to spinal neurons in organotypic slice culture. To assess the validity of this approach, we established serum‐free, defined‐medium organotypic cultures (DMOTC) from E13‐14 prenatal rats. Whole‐cell recordings were made from neurons maintained in DMOTC for up to 42 days. These were compared with recordings from neurons of similar age in acute spinal cord slices from 15‐ to 45‐day‐old rats. Five cell types were defined in acute slices as ‘Tonic’, ‘Irregular’, ‘Delay’, ‘Transient’ or ‘Phasic’ according to their discharge patterns in response to depolarizing current. Although fewer ‘Phasic’ cells were found in cultures, the proportions of ‘Tonic’, ‘Irregular’, ‘Delay’, and ‘Transient’ were similar to those found in acute slices. GABAergic, glycinergic, and ‘mixed’ inhibition were observed in neurons in acute slices and DMOTC. Pure glycinergic inhibition was absent in 7d cultures but became more pronounced as cultures aged. This parallels the development of glycinergic inhibition in vivo. These and other findings suggest that fundamental developmental processes related to neurotransmitter phenotype and neuronal firing properties are preserved in DMOTC. This validates their use in evaluating the cellular mechanisms that may contribute to the development of chronic pain.

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells. In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed.

FFAR3 (GPR41) is a G-protein coupled receptor for which short-chain fatty acids serve as endogenous ligands. The receptor is found on gut enteroendocrine L-cells, pancreatic β-cells, and sympathetic neurons, and is implicated in obesity, diabetes, allergic airway disease, and altered immune function. In primates, FFAR3 is segmentally duplicated resulting in GPR42, a gene currently classified as a suspected pseudogene. In this study, we sequenced FFAR3 and GPR42 open reading frames from 56 individuals and found an unexpectedly high frequency of polymorphisms contributing to several complex haplotypes. We also identified a frequent (18.8%) structural variation that results in GPR42 copy number polymorphism. Finally, sequencing revealed that 50.6% of GPR42 haplotypes differed from FFAR3 by only a single non-synonymous substitution and that the GPR42 reference sequence matched only 4.4% of the alleles. Sequencing of cDNA from human sympathetic ganglia and colon revealed processed transcripts matching the GPR42 genotype. Expression of several GPR42 haplotypes in rat sympathetic neurons revealed diverse pharmacological phenotypes that differed in potency and efficacy. Our data suggest that GPR42 be reclassified as a functioning gene and that recognition of sequence and copy number polymorphism of the FFAR3/GPR42 complex be considered during genetic and pharmacological investigation of these receptors.

LONG-TERM EFFECTS OF BRAIN-DERIVED NEUROTROPHIC FACTOR ON THE FREQUENCY OF INHIBITORY SYNAPTIC EVENTS IN THE RAT SUPERFICIAL DORSAL HORN

Chronic constriction injury (CCI) of rat sciatic nerve produces a specific pattern of electrophysiological changes in the superficial dorsal horn that lead to central sensitization that is associated with neuropathic pain. These changes can be recapitulated in spinal cord organotypic cultures by long term (5-6 days) exposure to brain-derived neurotrophic factor (BDNF) (200 ng/ml). Certain lines of evidence suggest that both CCI and BDNF increase excitatory synaptic drive to putative excitatory neurons while reducing that to putative inhibitory interneurons. Because BDNF slows the rate of discharge of synaptically-driven action potentials in inhibitory neurons, it should also decrease the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) throughout the superficial dorsal horn. To test this possibility, we characterized superficial dorsal horn neurons in organotypic cultures according to five electrophysiological phenotypes that included tonic, delay and irregular firing neurons. Five to 6 days of treatment with 200 ng/ml BDNF decreased sIPSC frequency in tonic and irregular neurons as might be expected if BDNF selectively decreases excitatory synaptic drive to inhibitory interneurons. The frequency of sIPSCs in delay neurons was however increased. Further analysis of the action of BDNF on tetrodotoxin-resistant miniature inhibitory postsynaptic currents (mIPSC) showed that the frequency was increased in delay neurons, unchanged in tonic neurons and decreased in irregular neurons. BDNF may thus reduce action potential frequency in those inhibitory interneurons that project to tonic and irregular neurons but not in those that project to delay neurons.

Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split.

RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel β subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Cavβ subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.

Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia

Rem2 is a member of the RGK subfamily of RAS small GTPases. Rem2 inhibits high voltage activated calcium channels, is involved in synaptogenesis, and regulates dendritic morphology. Rem2 is the primary RGK protein expressed in the nervous system, but to date, the precise expression patterns of this protein are unknown. In this study, we characterized Rem2 expression in the mouse nervous system. In the CNS, Rem2 mRNA was detected in all regions examined, but was enriched in the striatum. An antibody specific for Rem2 was validated using a Rem2 knockout mouse model and used to show abundant expression in striatonigral and striatopallidal medium spiny neurons but not in several interneuron populations. In the PNS, Rem2 was abundant in a subpopulation of neurons in the trigeminal and dorsal root ganglia, but was absent in sympathetic neurons of superior cervical ganglia. Under basal conditions, Rem2 was subject to post-translational phosphorylation, likely at multiple residues. Further, Rem2 mRNA and protein expression peaked at postnatal week two, which corresponds to the period of robust neuronal maturation in rodents. This study will be useful for elucidating the functions of Rem2 in basal ganglia physiology.

Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels

G-protein-coupled receptor modulation of voltage-gated ion channels is a common means of fine-tuning the response of channels to changes in membrane potential. Such modulation impacts physiological processes such as synaptic transmission, and hence therapeutic strategies often directly or indirectly target these pathways. As an exemplar of channel modulation, we examine strategies for investigating G-protein modulation of CaV2.2 or N-type voltage-gated Ca(2+) channels. We focus on biochemical and genetic tools for defining the molecular mechanisms underlying the various forms of CaV2.2 channel modulation initiated following ligand binding to G-protein-coupled receptors.