Swidbert R. Ott

Serotonin Mediates Behavioral Gregarization Underlying Swarm Formation in Desert Locusts

Desert locusts, Schistocerca gregaria, show extreme phenotypic plasticity, transforming between a little-seen solitarious phase and the notorious swarming gregarious phase depending on population density. An essential tipping point in the process of swarm formation is the initial switch from strong mutual aversion in solitarious locusts to coherent group formation and greater activity in gregarious locusts. We show here that serotonin, an evolutionarily conserved mediator of neuronal plasticity, is responsible for this behavioral transformation, being both necessary if behavioral gregarization is to occur and sufficient to induce it. Our data demonstrate a neurochemical mechanism linking interactions between individuals to large-scale changes in population structure and the onset of mass migration.

Gregarious desert locusts have substantially larger brains with altered proportions compared with the solitarious phase

The behavioural demands of group living and foraging have been implicated in both evolutionary and plastic changes in brain size. Desert locusts show extreme phenotypic plasticity, allowing brain morphology to be related to very different lifestyles in one species. At low population densities, locusts occur in a solitarious phase that avoids other locusts and is cryptic in appearance and behaviour. Crowding triggers the transformation into the highly active gregarious phase, which aggregates into dense migratory swarms. We found that the brains of gregarious locusts have very different proportions and are also 30 per cent larger overall than in solitarious locusts. To address whether brain proportions change with size through nonlinear scaling (allometry), we conducted the first comprehensive major axis regression analysis of scaling relations in an insect brain. This revealed that phase differences in brain proportions arise from a combination of allometric effects and deviations from the allometric expectation (grade shifts). In consequence, gregarious locusts had a larger midbrain∶optic lobe ratio, a larger central complex and a 50 per cent larger ratio of the olfactory primary calyx to the first olfactory neuropile. Solitarious locusts invest more in low-level sensory processing, having disproportionally larger primary visual and olfactory neuropiles, possibly to gain sensitivity. The larger brains of gregarious locusts prioritize higher integration, which may support the behavioural demands of generalist foraging and living in dense and highly mobile swarms dominated by intense intraspecific competition.

Timed and targeted differential regulation of nitric oxide synthase (NOS) and anti-NOS genes by reward conditioning leading to long-term memory formation

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene.

Confocal microscopy in large insect brains: Zinc–formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts

Confocal microscopy enables the analysis of immunofluorescence in whole-mount brains and is therefore widely used in the functional and comparative neuroanatomy of invertebrates. Three difficulties, however, are commonly encountered. First, poor penetration of antibodies after formaldehyde fixation impedes the immunostaining in central neuropile regions. Second, formaldehyde can cause a loss of antigenicity by epitope masking. Third, large brains must be cleared in hydrophobic media, a procedure that may distort morphology. I present a new methodology that overcomes these three problems by using zinc-formaldehyde (ZnFA) for fixation. The success of this technique is demonstrated in the brain of the desert locust and evaluated by comparison with fixation in formaldehyde and immunostaining against synapsin to reveal the regions of synaptic integration throughout the brain. ZnFA fixation markedly increased antibody penetration, prevented synapsin epitope masking, and in the cleared preparation the morphology of the brain was preserved with great fidelity. Possible mechanisms responsible for these improvements are discussed. Successful double labelling for synapsin and serotonin shows that small-molecule antigens are also retained by ZnFA fixation. The methodology should facilitate a range of applications including whole-mount brain stereology and the generation of digital standard brains. It may furthermore facilitate the detection of other protein antigens in large intact specimens such as vertebrate embryos.

Contralateral inhibition as a sensory bias: the neural basis for a female preference in a synchronously calling bushcricket, Mecopoda elongata.

Imperfect synchrony between male calls occurs widely in acoustically courting crickets and bushcrickets. Males which are able to establish the temporal leadership usually attract more females in choice experiments but the proximate mechanism for this precedence effect is unknown. Here we show that contralateral inhibition, the neural basis for lateral contrast enhancement in the auditory pathways of insects and vertebrates, is also the probable proximate neural mechanism for this female preference. We recorded simultaneously from a pair of identified auditory interneurons in the synchronizing bushcricket Mecopoda elongata. When two identical acoustic stimuli are presented from opposite directions, one preceding the other by 120 ms, the neural representation within the receiver is far stronger for the leader signal. This results from a suppression of the neural response to the follower chirp by reciprocal contralateral inhibition. The advantage of the representation of the leader is 2–3‐fold with time delays between 70 and 130 ms; the most clear‐cut female preferences have also been found with such delays in previous behavioural experiments. In time–intensity trading experiments, a lead by 120 ms could only be compensated for by increasing the amplitude of the follower signal by 7–11 dB. We discuss contralateral inhibition in auditory systems as a sensory bias that results in female preference for leading signals, with important evolutionary consequences for male calling strategies.

Microarray-based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts

Desert locusts (Schistocerca gregaria) show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST) database derived from S. gregaria central nervous system (CNS) to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.

Modeling Cooperative Volume Signaling in a Plexus of Nitric Oxide Synthase-Expressing Neurons

In vertebrate and invertebrate brains, nitric oxide (NO) synthase (NOS) is frequently expressed in extensive meshworks (plexuses) of exceedingly fine fibers. In this paper, we investigate the functional implications of this morphology by modeling NO diffusion in fiber systems of varying fineness and dispersal. Because size severely limits the signaling ability of an NO-producing fiber, the predominance of fine fibers seems paradoxical. Our modeling reveals, however, that cooperation between many fibers of low individual efficacy can generate an extensive and strong volume signal. Importantly, the signal produced by such a system of cooperating dispersed fibers is significantly more homogeneous in both space and time than that produced by fewer larger sources. Signals generated by plexuses of fine fibers are also better centered on the active region and less dependent on their particular branching morphology. We conclude that an ultrafine plexus is configured to target a volume of the brain with a homogeneous volume signal. Moreover, by translating only persistent regional activity into an effective NO volume signal, dispersed sources integrate neural activity over both space and time. In the mammalian cerebral cortex, for example, the NOS plexus would preferentially translate persistent regional increases in neural activity into a signal that targets blood vessels residing in the same region of the cortex, resulting in an increased regional blood flow. We propose that the fineness-dependent properties of volume signals may in part account for the presence of similar NOS plexus morphologies in distantly related animals.

Critical role for protein kinase A in the acquisition of gregarious behavior in the desert locust

The mechanisms that integrate genetic and environmental information to coordinate the expression of complex phenotypes are little understood. We investigated the role of two protein kinases (PKs) in the population density-dependent transition to gregarious behavior that underlies swarm formation in desert locusts: the foraging gene product, a cGMP-dependent PK (PKG) implicated in switching between alternative group-related behaviors in several animal species; and cAMP-dependent PK (PKA), a signal transduction protein with a preeminent role in different forms of learning. Solitarious locusts acquire key behavioral characters of the swarming gregarious phase within just 1 to 4 h of forced crowding. Injecting the PKA inhibitor KT5720 before crowding prevented this transition, whereas injecting KT5823, an inhibitor of PKG, did not. Neither drug altered the behavior of long-term gregarious locusts. RNAi against foraging effectively reduced its expression in the central nervous system, but this did not prevent gregarization upon crowding. By contrast, solitarious locusts with an RNAi-induced reduction in PKA catalytic subunit C1 expression behaved less gregariously after crowding, and RNAi against the inhibitory R1 subunit promoted more extensive gregarization following a brief crowding period. A central role of PKA is congruent with the recent discovery that serotonin mediates gregarization in locusts and with findings in vertebrates that similarly implicate PKA in the capacity to cope with adverse life events. Our results show that PKA has been coopted into effecting the wide-ranging transformation from solitarious to gregarious behavior, with PKA-mediated behavioral plasticity resulting in an environmentally driven reorganization of a complex phenotype.

Nitric oxide synthase in the thoracic ganglia of the locust: distribution in the neuropiles and morphology of neurones.

Nitric oxide signaling is implicated in olfactory and visual pathways within the insect brain. In contrast, little is known about the distribution and function of nitric oxide synthase (NOS) in the ventral nerve cord. This study uses NADPH diaphorase histochemistry to describe the anatomy of NOS‐containing neurones and the neuropilar distribution of NOS in the thoracic nerve cord of the locust. It is shown for the first time that mechanosensory neuropiles receive innervation from NOS‐containing interneurones. Different cells innervate exteroceptive and proprioceptive projection neuropiles. In the projection neuropiles of tactile afferents, a dense meshwork of NOS‐containing fibres is formed by collaterals of paired intersegmental axons that run through the entire thoracic nerve cord, innervating exclusively these exteroceptive neuropiles. In neuropile areas where proprioceptive afferents terminate, stained fibres are comparatively sparse and originate from local interneurones. The prothoracic ganglion showed strongly stained dense fibres in the dorsal neuropile that were not seen in the other neuromeres. This differential NOS‐expression can be related to the branching pattern of a ventral group of neurones that was different in each neuromere. All thoracic neuromeres and the abdominal neuromeres A2 and A3 of the metathoraic ganglion contained a previously undescribed type of unpaired median neurone with bilaterally ascending and descending intersegmental projections that stained strongly for NOS. The distribution of NOS found in this study suggests a novel role for nitric oxide in an early stage of mechanosensory information processing in all thoracic neuromeres and an additional role in the prothoracic ganglion, which might be related to behavioural specializations of the forelegs. J. Comp. Neurol. 395:217–230, 1998.

Nitric oxide synthase histochemistry in insect nervous systems: Methanol/formalin fixation reveals the neuroarchitecture of formaldehyde-sensitive NADPH diaphorase in the cockroach Periplaneta americana.

Formaldehyde‐insensitive NADPH diaphorase (NADPHd) activity is used widely as a histochemical marker for neuronal nitric oxide synthase (NOS). However, in several insects including the cockroach Periplaneta americana, NOS is apparently formaldehyde‐sensitive; NADPHd fails to reveal neuron morphology and results in faint generalized staining. Here we have used a novel fixative, methanol/ formalin (MF), to reveal for the first time the neuroarchitecture of NADPHd in the cockroach, with intense selective staining occurring in neurons throughout the brain and thoracic ganglia. Immunocytochemical and histochemical analysis of cockroach and locust nervous systems indicated that neuronal NADPHd after MF fixation can be attributed to NOS. However, NADPHd in locust glial and perineurial cells was histochemically different from that in neurons and may thus be due to enzymes other than NOS. Histochemical implications of species‐specific enzyme properties and of the transcriptional complexity of the NOS gene are discussed. The present findings suggest that MF fixation is a valuable new tool for the comparative analysis of the neuroarchitecture of NO signaling in insects. The Golgi‐like definition of the staining enabled analysis of the NADPHd architecture in the cockroach and comparison with that in the locust. NADPHd in the tactile neuropils of the thoracic ganglia showed a similar organization in the two species. The olfactory glomeruli of the antennal lobes were in both species densely innervated by NADPHd‐positive local interneurons that correlated in number with the number of glomeruli. Thus, the NADPHd architectures appear highly conserved in primary sensory neuropils. In the cockroach mushroom bodies, particularly dense staining in the γ‐layer of the lobes was apparently derived from Kenyon cells, whereas extrinsic arborizations were organized in domains across the lobes, an architecture that contrasts with the previously described tubular compartmentalization of locust mushroom bodies. These divergent architectures may result in different spatiotemporal dynamics of NO diffusion and suggest species differences in the role of NO in the mushroom bodies. J. Comp. Neurol. 448:165–185, 2002.