Individual Mesopontine Neurons Implicated in Anesthetic Loss-of-consciousness Employ Separate Ascending Pathways to the Cerebral Cortex.

The mesopontine tegmental anesthesia area (MPTA) is a small brainstem nucleus that, when exposed to minute quantities of GABAA receptor agonists, induces a state of general anesthesia. In addition to immobility and analgesia this state is accompanied by widespread suppression of neural activity in the cerebral cortex and high delta-band power in the electroencephalogram. Collectively, MPTA neurons are known to project to a variety of forebrain targets which are known to relay to the cortex in a highly distributed manner. Here we ask whether ascending projections of individual MPTA neurons collateralize to several of these cortical relay nuclei, or access only one. Using rats, contrasting retrograde tracers were microinjected pairwise on one side into three ascending relays: the basal forebrain, the zona incerta-lateral hypothalamus and the intralaminar thalamic nuclear group. In addition, in separate animals, each target was microinjected bilaterally. MPTA neurons were then identified as being single-or double-labeled, indicating projection to one target nucleus or collateralization to both. Results indicated that double-labeling was rare, occurring on average in only 1.3% of the neurons sampled. The overwhelming majority of individual MPTA neurons showed specific connectivity, contributing to only one of the major ascending pathways, either ipsilaterally or contralaterally, but not bilaterally. This architecture would permit particular functional aspects of anesthetic loss-of-consciousness to be driven by specific subpopulations of MPTA neurons.

Mesopontine Neurons Implicated in Anesthetic Loss-of-consciousness have Either Ascending or Descending Axonal Projections, but Not Both.

The MPTA (mesopontine tegmental anesthesia area) is a key node in a network of axonal pathways that collectively engage the key components of general anesthesia: immobility and atonia, analgesia, amnesia and loss-of-consciousness. In this study we have applied double retrograde tracing to analyze MPTA connectivity, with a focus on axon collateralization. Prior tracer studies have shown that collectively, MPTA neurons send descending projections to spinal and medullary brain targets associated with atonia and analgesia as well as ascending projections to forebrain structures associated with amnesia and arousal. Here we ask whether individual MPTA neurons collateralize broadly as might be expected of modulatory circuitry, sending axonal branches to both caudal and to rostral targets, or whether connectivity is more selective. Two distinguishable retrograde tracers were microinjected into pairs ("dyads") of known synaptic targets of the MPTA, one caudal and one rostral. We found that neurons that were double-labeled, and hence project to both targets were rare, constituting <0.5% on average of all MPTA neurons that project to these targets. The large majority sent axons either caudally, presumably to mediate mobility and/or antinociception, or rostrally, presumably to mediate mnemonic and/or arousal/cognitive functions. MPTA neurons with descending vs ascending projections also differed in size and shape, supporting the conclusion that they constitute distinct neuronal populations. From these and prior observations we conclude that the MPTA has a hybrid architecture including neurons with heterogeneous patterns of connectivity, some highly collateralized and some more targeted.

Model of anaesthetic induction by unilateral intracerebral microinjection of GABAergic agonists.

General anaesthetic agents induce loss of consciousness coupled with suppression of movement, analgesia and amnesia. Although these diverse functions are mediated by neural structures located in wide-ranging parts of the neuraxis, anaesthesia can be induced rapidly and reversibly by bilateral microinjection of minute quantities of γ-aminobutyric acid (GABA)A -R agonists at a small, focal locus in the mesopontine tegmentum (MPTA). State switching under these circumstances is presumably executed by dedicated neural pathways and does not require widespread distribution of the anaesthetic agent itself, the classical assumption regarding anaesthetic induction. Here it was asked whether these pathways serve each hemisphere independently, or whether there is bilateral redundancy such that the MPTA on each side is capable of anaesthetizing the entire brain. Either of two GABAA -R ligands were microinjected unilaterally into the MPTA in awake rats, the barbiturate modulator pentobarbital and the direct receptor agonist muscimol. Both agents, microinjected on either side, induced clinical anaesthesia, including bilateral atonia, bilateral analgesia and bilateral changes in cortical activity. The latter was monitored using c-fos expression and electroencephalography. This action, however, was not simply a consequence of suppressing spike activity in MPTA neurons, as unilateral (or bilateral) microinjection of the local anaesthetic lidocaine at the same locus failed to induce anaesthesia. A model of the state-switching circuitry that accounts for the bilateral action of unilateral microinjection and also for the observation that inactivation with lidocaine is not equivalent to inhibition with GABAA -R agonists was proposed. This is a step in defining the overall switching circuitry that underlies anaesthesia.

Expulsion of symbiotic algae during feeding by the green hydra--a mechanism for regulating symbiont density?

BACKGROUND: Algal-cnidarian symbiosis is one of the main factors contributing to the success of cnidarians, and is crucial for the maintenance of coral reefs. While loss of the symbionts (such as in coral bleaching) may cause the death of the cnidarian host, over-proliferation of the algae may also harm the host. Thus, there is a need for the host to regulate the population density of its symbionts. In the green hydra, Chlorohydra viridissima, the density of symbiotic algae may be controlled through host modulation of the algal cell cycle. Alternatively, Chlorohydra may actively expel their endosymbionts, although this phenomenon has only been observed under experimentally contrived stress conditions. PRINCIPAL FINDINGS: We show, using light and electron microscopy, that Chlorohydra actively expel endosymbiotic algal cells during predatory feeding on Artemia. This expulsion occurs as part of the apocrine mode of secretion from the endodermal digestive cells, but may also occur via an independent exocytotic mechanism. SIGNIFICANCE: Our results demonstrate, for the first time, active expulsion of endosymbiotic algae from cnidarians under natural conditions. We suggest this phenomenon may represent a mechanism whereby cnidarians can expel excess symbiotic algae when an alternative form of nutrition is available in the form of prey.

Osmotically driven prey disintegration in the gastrovascular cavity of the green hydra by a pore-forming protein.

Pore-forming proteins (PFPs) are water-soluble proteins able to integrate into target membranes to form transmembrane pores. They are common determinants of bacterial pathogenicity and are often found in animal venoms. We recently isolated and characterized Hydralysins (Hlns), paralytic PFPs from the venomous green hydra Chlorohydra viridissima that are not found within the nematocytes, suggesting they are not involved in prey capture. The present study aimed to decipher the biological role of Hlns. Using in situ hybridization and immunohistochemistry, we show that Hlns are expressed by digestive cells surrounding the gastrovascular cavity (GVC) of Chlorohydra and secreted onto the prey during feeding. At biologically relevant concentrations, Hlns bind prey membranes and form pores, lysing the cells and disintegrating the prey tissue. Hlns are unable to bind Chlorohydra membranes, thus protecting the producing animal from the destructive effect of its own cytolytic protein. We suggest that osmotic disintegration of the prey within the GVC by Hlns, followed by phagocytosis and intracellular digestion, allows the soft-bodied green hydra to feed on hard, cuticle-covered prey while lacking the physical means to mechanically disintegrate it. Our results extend the biological significance of PFPs beyond the commonly expected offensive or defensive roles.

Toxic polypeptides of the hydra--a bioinformatic approach to cnidarian allomones.

Cnidarians such as hydrae and sea anemones are sessile, predatory, soft bodied animals which depend on offensive and defensive allomones for prey capture and survival. These allomones are distributed throughout the entire organism both in specialized stinging cells (nematocytes) and in the body tissues. The cnidarian allomonal system is composed of neurotoxins, cytolysins and toxic phospholipapses. The present bioinformatic survey was motivated by the fact that while hydrae are the most studied model cnidarian, little is known about their allomones. A large-scale EST database from Hydra magnipapillata was searched for orthologs of known cnidarian allomones, as well as for allomones found in other venomous organisms. We show that the hydrae express orthologs of cnidarian phospholipase A2 toxins and cytolysins belonging to the actinoporin family, but could not find orthologs of the 'classic' short chain neurotoxins affecting sodium and potassium conductance. Hydrae also express proteins similar to elapid-like phospholipases, CRISP proteins, Prokineticin-like polypeptides and toxic deoxyribonucleases. Our results illustrate a high level of complexity in the hydra allomonal system, suggest that several toxins represent a basal component of all cnidarian allomones, and raise the intriguing possibility that similar proteins may fulfill both endogenous and allomonal roles in cnidaria.

Hydralysins, a new category of beta-pore-forming toxins in cnidaria.

Cnidaria are venomous animals that produce diverse protein and polypeptide toxins, stored and delivered into the prey through the stinging cells, the nematocytes. These include pore-forming cytolytic toxins such as well studied actinoporins. In this work, we have shown that the non-nematocystic paralytic toxins, hydralysins, from the green hydra Chlorohydra viridissima comprise a highly diverse group of beta-pore-forming proteins, distinct from other cnidarian toxins but similar in activity and structure to bacterial and fungal toxins. Functional characterization of hydralysins reveals that as soluble monomers they are rich in beta-structure, as revealed by far UV circular dichroism and computational analysis. Hydralysins bind erythrocyte membranes and form discrete pores with an internal diameter of approximately 1.2 nm. The cytolytic effect of hydralysin is cell type-selective, suggesting a specific receptor that is not a phospholipid or carbohydrate. Multiple sequence alignment reveals that hydralysins share a set of conserved sequence motifs with known pore-forming toxins such as aerolysin, epsilon-toxin, alpha-toxin, and LSL and that these sequence motifs are found in and around the poreforming domains of the toxins. The importance of these sequence motifs is revealed by the cloning, expression, and mutagenesis of three hydralysin isoforms that strongly differ in their hemolytic and paralytic activities. The correlation between the paralytic and cytolytic activities of hydralysin suggests that both are a consequence of receptor-mediated pore formation. Hydralysins and their homologues exemplify the wide distribution of beta-pore formers in biology and provide a useful model for the study of their molecular mode of action.

Hydralysin, a novel animal group-selective paralytic and cytolytic protein from a noncnidocystic origin in hydra.

In Cnidaria, the production of neurotoxic polypeptides is attributed to the ectodermal stinging cells (cnidocytes), which are discharged for offensive (prey capture) and/or defensive purposes. In this study, a new paralysis-inducing (neurotoxic) protein from the green hydra Chlorohydra viridissima was purified, cloned, and expressed. This paralytic protein is unique in that it (1) is derived from a noncnidocystic origin, (2) reveals a clear animal group-selective toxicity, (3) possesses an uncommon primary structure, remindful of pore-forming toxins, and (4) has a fast cytotoxic effect on insect cells but not on the tested mammalian cells. The possible biological role of such a noncnidocystic toxin is discussed.