Bruce A. Bamber

Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain.

Presynaptic terminals contain highly organized subcellular structures to facilitate neurotransmitter release. In C. elegans, the typical presynaptic terminal has an electron-dense active zone surrounded by synaptic vesicles. Loss-of-function mutations in the rpm-1 gene result in abnormally structured presynaptic terminals in GABAergic neuromuscular junctions (NMJs), most often manifested as a single presynaptic terminal containing multiple active zones. The RPM-1 protein has an RCC1-like guanine nucleotide exchange factor (GEF) domain and a RING-H2 finger. RPM-1 is most similar to the Drosophila presynaptic protein Highwire (HIW) and the mammalian Myc binding protein Pam. RPM-1 is localized to the presynaptic region independent of synaptic vesicles and functions cell autonomously. The temperature-sensitive period of rpm-1 coincides with the time of synaptogenesis. rpm-1 may regulate the spatial arrangement, or restrict the formation, of presynaptic structures.

The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor

Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion inCaenorhabditis elegans requires only theunc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure.unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently inXenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.

Presynaptic Terminals Independently Regulate Synaptic Clustering and Autophagy of GABAA Receptors in Caenorhabditis elegans

Synaptic clustering of GABAA receptors is important for the function of inhibitory synapses, influencing synapse strength and, consequently, the balance of excitation and inhibition in the brain. Presynaptic terminals are known to induce GABAA receptor clustering during synaptogenesis, but the mechanisms of cluster formation and maintenance are not known. To study how presynaptic neurons direct the formation of GABAA receptor clusters, we have investigated GABAA receptor localization in postsynaptic cells that fail to receive presynaptic contacts in Caenorhabditis elegans. Postsynaptic muscles in C. elegans receive acetylcholine and GABA motor innervation, and GABAA receptors cluster opposite GABA terminals. Selective loss of GABA inputs caused GABAA receptors to be diffusely distributed at or near the muscle cell surface, confirming that GABA presynaptic terminals induce GABAA receptor clustering. In contrast, selective loss of acetylcholine innervation had no effect on GABAA receptor localization. However, loss of both GABA and acetylcholine inputs together caused GABAA receptors to traffic to intracellular autophagosomes. Autophagosomes normally transport bulk cytoplasm to the lysosome for degradation. However, we show that GABAA receptors traffic to autophagosomes after endocytic removal from the cell surface and that acetylcholine receptors in the same cells do not traffic to autophagosomes. Thus, autophagy can degrade cell-surface receptors and can do so selectively. Our results show that presynaptic terminals induce GABAA receptor clustering by independently controlling synaptic localization and surface stability of GABAA receptors. They also demonstrate a novel function for autophagy in GABAA receptor degradative trafficking.

Three Distinct Amine Receptors Operating at Different Levels within the Locomotory Circuit Are Each Essential for the Serotonergic Modulation of Chemosensation in Caenorhabditis elegans

Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT6-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.

Monoamines and neuropeptides interact to inhibit aversive behaviour in Caenorhabditis elegans

Pain modulation is complex, but noradrenergic signalling promotes anti‐nociception, with α2‐adrenergic agonists used clinically. To better understand the noradrenergic/peptidergic modulation of nociception, we examined the octopaminergic inhibition of aversive behaviour initiated by the Caenorhabditis elegans nociceptive ASH sensory neurons. Octopamine (OA), the invertebrate counterpart of norepinephrine, modulates sensory‐mediated reversal through three α‐adrenergic‐like OA receptors. OCTR‐1 and SER‐3 antagonistically modulate ASH signalling directly, with OCTR‐1 signalling mediated by Gαo. In contrast, SER‐6 inhibits aversive responses by stimulating the release of an array of ‘inhibitory’ neuropeptides that activate receptors on sensory neurons mediating attraction or repulsion, suggesting that peptidergic signalling may integrate multiple sensory inputs to modulate locomotory transitions. These studies highlight the complexity of octopaminergic/peptidergic interactions, the role of OA in activating global peptidergic signalling cascades and the similarities of this modulatory network to the noradrenergic inhibition of nociception in mammals, where norepinephrine suppresses chronic pain through inhibitory α2‐adrenoreceptors on afferent nociceptors and stimulatory α1‐receptors on inhibitory peptidergic interneurons.

The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction

1 The unc‐49 gene of the nematode Caenorhabditis elegans encodes three γ‐aminobutyric acid type A (GABAA) receptor subunits. Two of these, UNC‐49B and UNC‐49C, are expressed at high abundance and co‐localize at the neuromuscular junction. 2 The UNC‐49B subunit is sufficient to form a GABAA receptor in vitro and in vivo. Furthermore, all loss‐of‐function unc‐49 alleles lack functional UNC‐49B. No mutations specifically inactivate UNC‐49C. Thus, UNC‐49C appears to be dispensable for receptor function; however, UNC‐49C has been conserved among different nematode species, suggesting it plays a necessary role. 3 To ascertain whether UNC‐49C is part of the GABAA receptor in vivo, we performed patch‐clamp electrophysiology on C. elegans muscle cells. Sensitivity to GABA, and to the antagonists picrotoxin and pregnenolone sulfate, matched the UNC‐49B/C heteromer rather than the UNC‐49B homomer, for both exogenous and synaptically‐released GABA. 4 The synaptic localization of UNC‐49C requires the presence of UNC‐49B, indicative of a physical association between the two subunits in vivo. Thus, the in vivo receptor is an UNC‐49B/C heteromer. 5 UNC‐49C plays a negative modulatory role. Using the rapid ligand‐exchange technique in vitro, we determined that UNC‐49C causes accelerated receptor desensitization. Previously, UNC‐49C was shown to reduce single‐channel conductance in UNC‐49B/C heteromers. Thus, the function of UNC‐49B is to provide GABA responsiveness and localization to synapses, while the function of UNC‐49C is to negatively modulate receptor function and precisely shape inhibitory postsynaptic currents.

Pharmacological characterization of the homomeric and heteromeric UNC-49 GABA receptors in C. elegans

UNC‐49B and UNC‐49C are γ‐aminobutyric acid (GABA) receptor subunits encoded by the Caenorhabditis elegans unc‐49 gene. UNC‐49B forms a homomeric GABA receptor, or can co‐assemble with UNC‐49C to form a heteromeric receptor. The pharmacological properties of UNC‐49B homomers and UNC‐49B/C heteromers were investigated in Xenopus oocytes. The UNC‐49 subunits are most closely related to the bicuculline‐ and benzodiazepine‐insensitive RDL GABA receptors of insects. Consistent with this classification, bicuculline (10 μM) did not inhibit, nor did diazepam (10 μM) enhance UNC‐49B homomeric or UNC‐49B/C heteromeric receptors. The UNC‐49C subunit strongly affects the pharmacology of UNC‐49B/C heteromeric receptors. UNC‐49B homomers were much more picrotoxin sensitive than UNC‐49B/C heteromers (IC50=0.9±0.2 μM and 166±42 μM, respectively). Pentobarbitone enhancement was greater for UNC‐49B homomers compared to UNC‐49B/C heteromers. Propofol (50 μM) slightly enhanced UNC‐49B homomers but slightly inhibited UNC‐49B/C heteromers. Penicillin G (10 mM) inhibited UNC‐49B homomers less strongly than UNC‐49B/C heteromers (30% compared to 53% inhibition, respectively). Several aspects of UNC‐49 pharmacology were unusual. Picrotoxin sensitivity strongly correlates with dieldrin sensitivity, yet UNC‐49B homomers were highly dieldrin resistant. The enhancing neurosteroid pregnanolone (5β‐pregnan‐3α‐ol‐20‐one; 10 μM) strongly inhibited both UNC‐49 receptors. Alphaxalone (10 μM), another enhancing neurosteroid, did not affect UNC‐49B homomers, but slightly inhibited UNC‐49B/C heteromers. UNC‐49 subunits and mammalian GABAA receptor α, β, and γ subunit classes all share roughly the same degree of sequence similarity. Thus, although they are most similar to other invertebrate GABA receptors, the UNC‐49 receptors share significant structural and pharmacological overlap with mammalian GABAA receptors.

Residues in the first transmembrane domain of the Caenorhabditis elegans GABAA receptor confer sensitivity to the neurosteroid pregnenolone sulfate

1 The GABAA receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABAA receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC‐49 GABA receptor, a close homolog of the mammalian GABAA receptor. 2 The UNC‐49 locus encodes two GABA receptor subunits, UNC‐49B and UNC‐49C. UNC‐49C is sensitive to PS but UNC‐49B is not sensitive. By analyzing chimeric receptors and receptors containing site‐directed mutations, we identified two regions required for PS inhibition. 3 Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. 4 Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.

Serotonin differentially modulates Ca2+ transients and depolarization in a C. elegans nociceptor

Monoamines and neuropeptides modulate neuronal excitability and synaptic strengths, shaping circuit activity to optimize behavioral output. In C. elegans, a pair of bipolar polymodal nociceptors, the ASHs, sense 1-octanol to initiate escape responses. In the present study, 1-octanol stimulated large increases in ASH Ca(2+), mediated by L-type voltage-gated Ca(2+) channels (VGCCs) in the cell soma and L-plus P/Q-type VGCCs in the axon, which were further amplified by Ca(2+) released from intracellular stores. Importantly, 1-octanol-dependent aversive responses were not inhibited by reducing ASH L-VGCC activity genetically or pharmacologically. Serotonin, an enhancer of 1-octanol avoidance, potentiated 1-octanol-dependent ASH depolarization measured electrophysiologically, but surprisingly, decreased the ASH somal Ca(2+) transients. These results suggest that ASH somal Ca(2+) transient amplitudes may not always be predictive of neuronal depolarization and synaptic output. Therefore, although increases in steady-state Ca(2+) can reliably indicate when neurons become active, quantitative relationships between Ca(2+) transient amplitudes and neuronal activity may not be as straightforward as previously anticipated.

Shaping cellular form and function by autophagy.

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the g-aminobutyric acid type A receptor (GABAA receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function. Addendum to: Presynaptic Terminals Independently Regulate Synaptic Clustering and Autophagy of GABAA Receptors in Caenorhabditis elegans .A.M. Rowland, J.E. Richmond, J.G. Olsen, D.H. Hall and B. A. Bamber J Neurosci 2006; 26:1711-20