Shannon Meisner

Spider Peripheral Mechanosensory Neurons Are Directly Innervated and Modulated by Octopaminergic Efferents

Octopamine is a chemical relative of noradrenaline providing analogous neurohumoral control of diverse invertebrate physiological processes. There is also evidence for direct octopaminergic innervation of some insect peripheral tissues. Here, we show that spider peripheral mechanoreceptors are innervated by octopamine-containing efferents. The mechanosensory neurons have octopamine receptors colocalized with synapsin labeling in the efferent fibers. In addition, octopamine enhances the electrical response of the sensory neurons to mechanical stimulation. Spider peripheral mechanosensilla receive extensive efferent innervation. Many efferent fibers in the legs of Cupiennius salei are GABAergic, providing inhibitory control of sensory neurons, but there is also evidence for other neurotransmitters. We used antibody labeling to show that some efferents contain octopamine and that octopamine receptors are concentrated on the axon hillocks and proximal soma regions of all mechanosensory neurons in the spider leg. Synaptic vesicles in efferent neurons were concentrated in similar areas. Octopamine, or its precursor tyramine, increased responses of mechanically stimulated filiform (trichobothria) leg hairs. This effect was blocked by the octopamine antagonist phentolamine. The octopamine-induced modulation was mimicked by 8-Br-cAMP, a cAMP analog, and blocked by Rp-cAMPS, a protein kinase A inhibitor, indicating that spider octopamine receptors activate adenylate cyclase and increase cAMP concentration. Frequency response analysis showed that octopamine increased the sensitivity of the trichobothria neurons over a broad frequency range. Thus, the major effect of octopamine is to increase its overall sensitivity to wind-borne signals from sources such as flying insect prey or predators.

Dendritic excitability and localization of GABA-mediated inhibition in spider mechanoreceptor neurons.

GABAergic inhibition of mechanosensory afferent axon terminals is a widespread phenomenon in vertebrates and invertebrates. Spider mechanoreceptor neurons receive efferent innervation on their peripherally located axons, somata and sensory dendrites, and the dendrites have recently been shown to be excitable. Excitability of the spider sensory neurons is inhibited by muscimol and GABA, agonists of ionotropic GABA receptors. Here we asked where in the neurons this inhibition occurs. We found no evidence for inhibition of action potentials in the sensory dendrites, but axonal action potentials were rapidly suppressed by both agonists. Earlier work showed that metabotropic GABAB receptors are located on the dendrites and distal somata of the spider sensory neurons, where they modulate voltage‐activated conductances and may provide slower, prolonged inhibition. Therefore, GABA released from single peripheral efferents may activate both ionotropic and metabotropic receptor types, providing rapid suppression of axonal activity followed by slower inhibition that eventually prevents action potential initiation in the distal dendrites.

Ca2+/calmodulin-dependent protein kinase II mediates the octopamine-induced increase in sensitivity in spider VS-3 mechanosensory neurons

G‐protein‐coupled octopamine (OA) receptors mediate their effects by Ca2+ signaling or adjusting intracellular cAMP levels. Depending on OA concentration and cell type, activation of OA receptors in excitable cells triggers excitatory or inhibitory effects, but the mechanisms by which Ca2+ or cAMP mediates these effects are not well understood. We investigated signaling mechanisms that are potentially activated by OA, and OA effects on excitability and frequency sensitivity in mechanosensory neurons innervating the VS‐3 slit sensilla on the patella of the spider Cupiennius salei. These neurons are directly innervated by octopaminergic efferents, and possess OA receptors that were immunoreactive to an antibody against an OA receptor highly expressed in mushroom bodies. OA application enhanced VS‐3 neuron sensitivity, especially at high stimulation frequencies. This enhancement lasted for at least 1 h after OA application. Changes in sensitivity were also detected when the Ca2+ ionophore ionomycin or the cAMP analog 8‐Br‐cAMP was applied. However, the cAMP pathway was unlikely to mediate the OA effect, as the protein kinase A inhibitor RP‐cAMPS did not diminish this effect. In contrast, the OA‐induced sensitivity enhancement was significantly reduced by KN‐62, an inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaMKII), and by the Ca2+ chelator BAPTA‐AM. OA depolarized the neurons by 3.8 mV from resting potential, well below the threshold for opening of voltage‐activated Ca2+ channels. OA also reduced the amplitudes of voltage‐activated K+ currents. We propose that OA receptors in VS‐3 neurons activate inositol 1,4,5‐trisphosphate, leading to Ca2+ release from intracellular stores. The Ca2+ surge switches on CaMKII, which modulates voltage‐activated K+ channels, resulting in persistent enhancement in excitability.

Dynamic properties of Drosophila olfactory electroantennograms

Time-dependent properties of chemical signals are probably crucially important to many animals, but little is known about the dynamics of chemoreceptors. Behavioral evidence of dynamic sensitivity includes the control of moth flight by pheromone plume structure, and the ability of some blood-sucking insects to detect varying concentrations of carbon dioxide, possibly matched to host breathing rates. Measurement of chemoreceptor dynamics has been limited by the technical challenge of producing controlled, accurate modulation of olfactory and gustatory chemical concentrations over suitably wide ranges of amplitude and frequency. We used a new servo-controlled laminar flow system, combined with photoionization detection of surrogate tracer gas, to characterize electroantennograms (EAG) of Drosophila antennae during stimulation with fruit odorants or aggregation pheromone in air. Frequency response functions and coherence functions measured over a bandwidth of 0–100 Hz were well characterized by first-order low-pass linear filter functions. Filter time constant varied over almost a tenfold range, and was characteristic for each odorant, indicating that several dynamically different chemotransduction mechanisms are present. Pheromone response was delayed relative to fruit odors. Amplitude of response, and consequently signal-to-noise ratio, also varied consistently with different compounds. Accurate dynamic characterization promises to provide important new information about chemotransduction and odorant-stimulated behavior.

Transcriptome analysis and RNA interference of cockroach phototransduction indicate three opsins and suggest a major role for TRPL channels

Our current understanding of insect phototransduction is based on a small number of species, but insects occupy many different visual environments. We created the retinal transcriptome of a nocturnal insect, the cockroach, Periplaneta americana to identify proteins involved in the earliest stages of compound eye phototransduction, and test the hypothesis that different visual environments are reflected in different molecular contributions to function. We assembled five novel mRNAs: two green opsins, one UV opsin, and one each TRP and TRPL ion channel homologs. One green opsin mRNA (pGO1) was 100–1000 times more abundant than the other opsins (pGO2 and pUVO), while pTRPL mRNA was 10 times more abundant than pTRP, estimated by transcriptome analysis or quantitative PCR (qPCR). Electroretinograms were used to record photoreceptor responses. Gene-specific in vivo RNA interference (RNAi) was achieved by injecting long (596–708 bp) double-stranded RNA into head hemolymph, and verified by qPCR. RNAi of the most abundant green opsin reduced both green opsins by more than 97% without affecting UV opsin, and gave a maximal reduction of 75% in ERG amplitude 7 days after injection that persisted for at least 19 days. RNAi of pTRP and pTRPL genes each specifically reduced the corresponding mRNA by 90%. Electroretinogram (ERG) reduction by pTRPL RNAi was slower than for opsin, reaching 75% attenuation by 21 days, without recovery at 29 days. pTRP RNAi attenuated ERG much less; only 30% after 21 days. Combined pTRP plus pTRPL RNAi gave only weak evidence of any cooperative interactions. We conclude that silencing retinal genes by in vivo RNAi using long dsRNA is effective, that visible light transduction in Periplaneta is dominated by pGO1, and that pTRPL plays a major role in cockroach phototransduction.

Acetylcholine receptors in spider peripheral mechanosensilla

Peripherally located parts of spider mechanosensory neurons are modulated by several neurotransmitters released from apposed efferent fibers. Activities of acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) and ACh degrading enzyme acetylcholine esterase (AChE) were previously found in some efferent fibers. ChAT activity was also present in all the mechanosensory neurons, while AChE activity was only found in some. We show that spider mechanosensory neurons and probably some efferent neurons are immunoreactive to a monoclonal antibody against muscarinic ACh receptors (mAChRs). However, application of muscarinic agonists did not change the physiological responses or membrane potentials of neurons in the lyriform organ VS-3. Similarly, the sensitivities of the neurons of trichobothria (filiform hairs) remained unchanged after application of these agonists. Therefore, activation of mAChRs may only modulate the function of spider mechanosensory neurons indirectly, for example, by affecting the release of other transmitter(s). However, a subgroup of VS-3 neurons was inhibited by ACh, which also depolarized the membrane similar to these neurons’ responses to GABA, suggesting that ACh activates anion channels in these neurons. Interestingly, all of the neurons responding to ACh were the rapidly adapting Type A neurons that were previously shown to express AChE activity.

GABA and glutamate receptors have different effects on excitability and are differentially regulated by calcium in spider mechanosensory neurons

GABA and glutamate receptors belonging to the ligand‐gated chloride‐channel family are primary targets of insecticides and antiparasitics, so their molecular structure, pharmacology and biophysical properties have attracted significant attention. However, little is known about the physiological roles of these channels or how they regulate neuronal excitability and animal behavior. Mechanosensory neurons of VS‐3 slit sensilla in the patella of the tropical wandering spider, Cupiennius salei, react to the GABAA‐receptor agonists, GABA and muscimol, with depolarization and an increase in intracellular [Ca2+] and, during random noise stimulation, with a mixed inhibitory–excitatory response. We established that the GABAA‐receptors in all VS‐3 neurons are identical, but there are at least two types of glutamate receptors and some neurons do not respond to glutamate at all. Immunohistochemistry with antibodies against Drosophila inhibitory glutamate receptor (GluCls) α‐subunit suggests that in addition to VS‐3 neurons, these receptors may also be present in the efferent neurons surrounding the sensory neurons. Most VS‐3 neurons were inhibited but not depolarized by glutamate during random stimulation, but some depolarized and had a similar excitatory–inhibitory response to glutamate as to muscimol. The membrane‐permeable Ca2+‐chelator BAPTA‐AM abolished muscimol effects but potentiated glutamate effects, indicating that GABA and glutamate receptors are differentially modulated by Ca2+, leading to diverse regulation of neuronal excitability. We hypothesize that this could be achieved by different Ca2+‐triggered phosphorylation processes at each receptor type. These findings are important for understanding the significance of Ca2+‐mediated regulation of transmitter receptor molecules and its role in controlling excitability.

Immunocytochemical localization of choline acetyltransferase and muscarinic ACh receptors in the antenna during development of the sphinx moth Manduca sexta.

Immunocytochemistry with monoclonal antibodies was used to investigate the locations of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) in sections of the developing antennae of the moth Manduca sexta. The results were correlated with a previous morphological investigation in the developing antennae which allowed us to locate different cell types at various stages of development. Our findings indicated that the muscarinic cholinergic system was not restricted to the sensory neurons but was also present in glial and epidermal cells. By day 4–5 of adult development, immunoreactivity against both antibodies was present in the axons of the antennal nerve, and more intense labeling was present in sections from older pupae. At days 4–9, the cell bodies of the sensory neurons in the basal part of the epidermis were also intensely immunolabeled by the anti-mAChR antibody. In mature flagella, large numbers of cells, some with processes into hairs, were strongly labeled by both antibodies. Antennal glial cells were intensely immunolabeled with both antibodies by days 4–5, but in later stages, it was not possible to discriminate between glial and neural staining. At days 4–9, we observed a distinctly labeled layer of epidermal cells close to the developing cuticle. The expression of both ChAT and mAChRs by neurons in moth antennae may allow the regulation of excitability by endogenous ACh. Cholinergic communication between neurons and glia may be part of the system that guides axon elongation during development. The cholinergic system in the apical part of the developing epidermis could be involved in cuticle formation.

Co-localization of Gamma-Aminobutyric Acid and Glutamate in Neurons of the Spider Central Nervous System

Spider sensory neurons with cell bodies close to various sensory organs are innervated by putative efferent axons from the central nervous system (CNS). Light and electronmicroscopic imaging of immunolabeled neurons has demonstrated that neurotransmitters present at peripheral synapses include γ-aminobutyric acid (GABA), glutamate and octopamine. Moreover, electrophysiological studies show that these neurotransmitters modulate the sensitivity of peripheral sensory neurons. Here, we undertook immunocytochemical investigations to characterize GABA and glutamate-immunoreactive neurons in three-dimensional reconstructions of the spider CNS. We document that both neurotransmitters are abundant in morphologically distinct neurons throughout the CNS. Labeling for the vesicular transporters, VGAT for GABA and VGLUT for glutamate, showed corresponding patterns, supporting the specificity of antibody binding. Whereas some neurons displayed strong immunolabeling, others were only weakly labeled. Double labeling showed that a subpopulation of weakly labeled neurons present in all ganglia expresses both GABA and glutamate. Double labeled, strongly and weakly labeled GABA and glutamate immunoreactive axons were also observed in the periphery along muscle fibers and peripheral sensory neurons. Electron microscopic investigations showed presynaptic profiles of various diameters with mixed vesicle populations innervating muscle tissue as well as sensory neurons. Our findings provide evidence that: (1) sensory neurons and muscle fibers are innervated by morphologically distinct, centrally located GABA- and glutamate immunoreactive neurons; (2) a subpopulation of these neurons may co-release both neurotransmitters; and (3) sensory neurons and muscles are innervated by all of these neurochemically and morphologically distinct types of neurons. The biochemical diversity of presynaptic innervation may contribute to how spiders filter natural stimuli and coordinate appropriate response patterns.

Carbon dioxide and fruit odor transduction in Drosophila olfactory neurons. What controls their dynamic properties

We measured frequency response functions between odorants and action potentials in two types of neurons in Drosophila antennal basiconic sensilla. CO2 was used to stimulate ab1C neurons, and the fruit odor ethyl butyrate was used to stimulate ab3A neurons. We also measured frequency response functions for light-induced action potential responses from transgenic flies expressing H134R-channelrhodopsin-2 (ChR2) in the ab1C and ab3A neurons. Frequency response functions for all stimulation methods were well-fitted by a band-pass filter function with two time constants that determined the lower and upper frequency limits of the response. Low frequency time constants were the same in each type of neuron, independent of stimulus method, but varied between neuron types. High frequency time constants were significantly slower with ethyl butyrate stimulation than light or CO2 stimulation. In spite of these quantitative differences, there were strong similarities in the form and frequency ranges of all responses. Since light-activated ChR2 depolarizes neurons directly, rather than through a chemoreceptor mechanism, these data suggest that low frequency dynamic properties of Drosophila olfactory sensilla are dominated by neuron-specific ionic processes during action potential production. In contrast, high frequency dynamics are limited by processes associated with earlier steps in odor transduction, and CO2 is detected more rapidly than fruit odor.