Riding on the wind: volatile compounds dictate selection of grassland seedlings by snails.

Background and Aims: Seedling herbivory is an important selective filter in many plant communities. The removal of preferred food plants by both vertebrate and, more commonly, invertebrate herbivores can destroy entire seedling cohorts, and consequently dictate plant community assembly. Nevertheless, our understanding of how and why some seedlings are more prone to herbivore attack than their neighbours remains limited. For seedlings, where even minor tissue damage is fatal, avoiding contact with herbivores is probably advantageous and, on this basis, volatile organic compounds (VOCs) are strong candidates to fulfil a primary defensive role. Methods: We quantified seedling selection by snails (Cornu aspersum) for 14 common, European grassland species. Seedling acceptability was subsequently compared with species-specific expression of constitutive secondary defence metabolites (CSDMs), and VOCs to determine their relative influence on seedling selection. Results: We found no relationship between seedling acceptability and CSDMs, but seedling selection was strongly associated with VOC profiles. Monoterpenes (specifically ß-ocimene) were identified as likely attractants, while green leaf volatiles (GLVs) (3-hexen-1-ol acetate) were strongly associated with low seedling acceptability. Conclusions: By elucidating a relationship between VOCs and seedling acceptability, we contradict a long-held, but poorly tested, assumption that seedling selection by herbivores in (semi-)natural plant communities centres on CSDMs. Instead, our results corroborate recent work showing how GLVs, including 3-hexen-1-ol acetate, deter crop seedling selection by molluscs. Although our failure to establish any early-ontogenetic relationship between VOCs and CSDMs also suggests that the former do not 'advertise' possession of the latter, we nevertheless reveal the role that VOCs play in defending seedlings against herbivory before lethal damage occurs.

Something in the air? The impact of volatiles on mollusc attack of oilseed rape seedlings.

BACKGROUND AND AIMS: Mounting concerns about balancing food security with the environmental impacts of agro-chemical use underpin the need to better understand the mechanisms by which crop plants, particularly during the vulnerable seedling stage, attract or repel herbivores. METHODS: The feeding preferences of the mollusc Helix aspersa were determined for several oilseed rape (Brassica napus) cultivars and a rank order of acceptability was established. This was compared with glucosinolate concentrations and volatile organic compound (VOC) profiles to determine whether seedling acceptability to molluscs was linked to either form of defence. KEY RESULTS: While VOC profiles for each oilseed rape cultivar could be separated by canonical discriminant analysis and associated with mollusc feeding preferences, glucosinolate profiles were unrelated to snail feeding behaviour. A mixture of monoterpenes (α-pinene, ß-myrcene and δ-3-carene) was identified as a putative attractant, while a blend of the green leaf volatiles 3-hexen-1-ol, 3-hexen-1-ol acetate and the monoterpene α-terpinene was identified as a putative repellent mix. Added to the VOC profile of oilseed rape seedlings, the 'repellent' mix reduced mollusc selection, while the 'attractant' mix had no effect. CONCLUSIONS: Despite the widespread assumption that seedling selection by generalist herbivores is governed by chemical defence and taste, we show that olfactory cues may be more important. Oilseed rape may be atypical of wild plants, but our ability to identify repellent volatile organic compounds that can influence snail olfactory selection points to new methods for crop protection using modified VOC profiles during the vulnerable seedling stage.

Functional connectivity in raphé-pontomedullary circuits supports active suppression of breathing during hypocapnic apnea.

Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04-0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity.

Pontine respiratory-modulated activity before and after vagotomy in decerebrate cats.

The dorsolateral (DL) pons modulates the respiratory pattern. With the prevention of lung inflation during central inspiratory phase (no-inflation (no-I or delayed-I) tests), DL pontine neuronal activity increased the strength and consistency of its respiratory modulation, properties measured statistically by the eta(2) value. This increase could result from enhanced respiratory-modulated drive arising from the medulla normally gated by vagal activity. We hypothesized that DL pontine activity during delayed-I tests would be comparable to that following vagotomy. Ensemble recordings of neuronal activity were obtained before and after vagotomy and during delayed-I tests in decerebrate, paralysed and ventilated cats. In general, changes in activity pattern during the delayed-I tests were similar to those after vagotomy, with the exception of firing-rate differences at the inspiratory-expiratory phase transition. Even activity that was respiratory-modulated with the vagi intact became more modulated while withholding lung inflation and following vagotomy. Furthermore, we recorded activity that was excited by lung inflation as well as changes that persisted past the stimulus cycle. Computer simulations of a recurrent inhibitory neural network model account not only for enhanced respiratory modulation with vagotomy but also the varied activities observed with the vagi intact. We conclude that (a) DL pontine neurones receive both vagal-dependent excitatory inputs and central respiratory drive; (b) even though changes in pontine activity are transient, they can persist after no-I tests whether or not changes in the respiratory pattern occur in the subsequent cycles; and (c) models of respiratory control should depict a recurrent inhibitory circuitry, which can act to maintain the stability and provide plasticity to the respiratory pattern.

Arterial pulse modulated activity is expressed in respiratory neural output.

Although it is well-established that sympathetic activity is modulated with respiration, it is unknown whether neural control of respiration is reciprocally influenced by cardiovascular function. Even though previous studies have suggested the existence of pulse modulation in respiratory neurons, they could not exclude the possibility that such cells were involved in cardiovascular rather than respiratory motor control, owing to neuroanatomic and functional overlaps between brain stem neurons involved in respiratory and cardiovascular control. The aim of this study was to test the hypothesis that respiratory motoneurons and putative premotoneurons are modulated by arterial pulse. An existing data set composed of 72 well-characterized, respiratory-modulated brain stem motoneurons and putative premotoneurons was analyzed using delta(2), a recently described statistic that quantifies the magnitude of arterial pulse-modulated spike activity [Dick TE and Morris KF. J Physiol 556: 959-970, 2004]. Neuronal activity was recorded in the rostral and caudal ventral respiratory groups of 19 decerebrate, neuromuscular-blocked, ventilated cats. Axonal projections were identified by rectified and unrectified spike-triggered averages of recurrent laryngeal nerve activity or by antidromic activation from spinal stimulation electrodes. The firing rates of approximately 30% of these neurons were modulated in phase with both the respiratory and cardiac cycles. Furthermore, arterial pulse modulation occurred preferentially in the expiratory phase in that only expiratory neurons had high delta(2) values and only expiratory activity had significant delta(2) values after partitioning tonic activity into the inspiratory and expiratory phases. The results demonstrate that both respiratory motoneurons and putative premotoneuronal activity can be pulse modulated. We conclude that a cardiac cycle-related modulation is expressed in respiratory motor activity, complementing the long-recognized respiratory modulation of sympathetic nerve activity.

Ventrolateral medullary respiratory network participation in the expiration reflex in the cat.

The expiration reflex is a distinct airway defensive response characterized by a brief, intense expiratory effort and coordinated adduction and abduction of the laryngeal folds. This study addressed the hypothesis that the ventrolateral medullary respiratory network participates in the reflex. Extracellular neuron activity was recorded with microelectrode arrays in decerebrated, neuromuscular-blocked, ventilated cats. In 32 recordings (17 cats), 232 neurons were monitored in the rostral (including Bötzinger and pre-Bötzinger complexes) and caudal ventral respiratory group. Neurons were classified by firing pattern, evaluated for spinal projections, functional associations with recurrent laryngeal and lumbar nerves, and firing rate changes during brief, large increases in lumbar motor nerve discharge (fictive expiration reflex, FER) elicited during mechanical stimulation of the vocal folds. Two hundred eight neurons were respiratory modulated, and 24 were nonrespiratory; 104 of the respiratory and 6 of the nonrespiratory-modulated neurons had altered peak firing rates during the FER. Increased firing rates of bulbospinal neurons and expiratory laryngeal premotor and motoneurons during the expiratory burst of FER were accompanied by changes in the firing patterns of putative propriobulbar neurons proposed to participate in the eupneic respiratory network. The results support the hypothesis that elements of the rostral and caudal ventral respiratory groups participate in generating and shaping the motor output of the FER. A model is proposed for the participation of the respiratory network in the expiration reflex.

Medullary raphe neuron activity is altered during fictive cough in the decerebrate cat.

Chemical lesions in the medullary raphe nuclei region influence cough. This study examined whether firing patterns of caudal medullary midline neurons were altered during cough. Extracellular neuron activity was recorded with microelectrode arrays in decerebrated, neuromuscular-blocked, ventilated cats. Cough-like motor patterns (fictive cough) in phrenic and lumbar nerves were elicited by mechanical stimulation of the intrathoracic trachea. Discharge patterns of respiratory and nonrespiratory-modulated neurons were altered during cough cycles (58/133); 45 increased and 13 decreased activity. Fourteen cells changed firing rate during the inspiratory and/or expiratory phases of cough. Altered patterns in 43 cells were associated with the duration of, or extended beyond, the cough episodes. The different response categories suggest that multiple factors influence the discharge patterns during coughing: e.g., respiratory-modulated and tonic inputs and intrinsic connections. These results suggest involvement of midline neurons (i.e., raphe nuclei) in the cough reflex.

Pontine respiratory group neuron discharge is altered during fictive cough in the decerebrate cat.

A network of neurons in the rostral dorsal lateral pons and pons/mescencephalic junction constitute the pontine respiratory group (PRG) and is essential for reflex cough. As a next step in understanding the role of the PRG in the expression of the cough reflex, we examined neuron firing rates during fictive cough in cats. Decerebrated, thoracotomized, paralyzed, cycle-triggered ventilated adult cats were used. Extracellular activity of many single neurons and phrenic and lumbar neurograms were monitored during fictive cough produced by mechanical stimulation of the intrathoracic trachea. Neurons were tested during control periods for respiratory modulation of firing rate by cycle-triggered histograms and statistical tests. Most respiratory modulated cells were continuously active with various superimposed respiratory patterns; major categories included inspiratory decrementing (I-Dec), expiratory decrementing (E-Dec) and expiratory augmenting (E-Aug). There were alterations in the discharge patterns of respiratory, as well as, non-respiratory modulated neurons during cough. The results suggest an involvement of the PRG in the configuration of the cough motor pattern.

The effects of natural iron fertilisation on deep-sea ecology: the Crozet Plateau, Southern Indian Ocean.

The addition of iron to high-nutrient low-chlorophyll (HNLC) oceanic waters stimulates phytoplankton, leading to greater primary production. Large-scale artificial ocean iron fertilization (OIF) has been proposed as a means of mitigating anthropogenic atmospheric CO(2), but its impacts on ocean ecosystems below the photic zone are unknown. Natural OIF, through the addition of iron leached from volcanic islands, has been shown to enhance primary productivity and carbon export and so can be used to study the effects of OIF on life in the ocean. We compared two closely-located deep-sea sites (∼400 km apart and both at ∼4200 m water depth) to the East (naturally iron fertilized; +Fe) and South (HNLC) of the Crozet Islands in the southern Indian Ocean. Our results suggest that long-term geo-engineering of surface oceanic waters via artificial OIF would lead to significant changes in deep-sea ecosystems. We found that the +Fe area had greater supplies of organic matter inputs to the seafloor, including polyunsaturated fatty acid and carotenoid nutrients. The +Fe site also had greater densities and biomasses of large deep-sea animals with lower levels of evenness in community structuring. The species composition was also very different, with the +Fe site showing similarities to eutrophic sites in other ocean basins. Moreover, major differences occurred in the taxa at the +Fe and HNLC sites revealing the crucial role that surface oceanic conditions play in changing and structuring deep-sea benthic communities.

Central and peripheral chemoreceptors evoke distinct responses in simultaneously recorded neurons of the raphé-pontomedullary respiratory network.

The brainstem network for generating and modulating the respiratory motor pattern includes neurons of the medullary ventrolateral respiratory column (VRC), dorsolateral pons (PRG) and raphé nuclei. Midline raphé neurons are proposed to be elements of a distributed brainstem system of central chemoreceptors, as well as modulators of central chemoreceptors at other sites, including the retrotrapezoid nucleus. Stimulation of the raphé system or peripheral chemoreceptors can induce a long-term facilitation of phrenic nerve activity; central chemoreceptor stimulation does not. The network mechanisms through which each class of chemoreceptor differentially influences breathing are poorly understood. Microelectrode arrays were used to monitor sets of spike trains from 114 PRG, 198 VRC and 166 midline neurons in six decerebrate vagotomized cats; 356 were recorded during sequential stimulation of both receptor classes via brief CO(2)-saturated saline injections in vertebral (central) and carotid arteries (peripheral). Seventy neurons responded to both stimuli. More neurons were responsive only to peripheral challenges than those responsive only to central chemoreceptor stimulation (PRG, 20 : 4; VRC, 41 : 10; midline, 25 : 13). Of 16 474 pairs of neurons evaluated for short-time scale correlations, similar percentages of reference neurons in each brain region had correlation features indicative of a specific interaction with at least one target neuron: PRG (59.6%), VRC (51.0%) and raphé nuclei (45.8%). The results suggest a brainstem network architecture with connectivity that shapes the respiratory motor pattern via overlapping circuits that modulate central and peripheral chemoreceptor-mediated influences on breathing.

Pontine-ventral respiratory column interactions through raphe circuits detected using multi-array spike train recordings.

Recently, Segers et al. identified functional connectivity between the ventrolateral respiratory column (VRC) and the pontine respiratory group (PRG). The apparent sparseness of detected paucisynaptic interactions motivated consideration of other potential functional pathways between these two regions. We report here evidence for "indirect" serial functional linkages between the PRG and VRC via intermediary brain stem midline raphé neurons. Arrays of microelectrodes were used to record sets of spike trains from a total of 145 PRG, 282 VRC, and 340 midline neurons in 11 decerebrate, vagotomized, neuromuscularly blocked, ventilated cats. Spike trains of 13,843 pairs of neurons that included at least one raphé cell were screened for respiratory modulation and short-time scale correlations. Significant correlogram features were detected in 7.2% of raphé-raphé (291/4,021), 4.3% of VRC-raphé (292/6,755), and 4.0% of the PRG-raphé (124/3,067) neuron pairs. Central peaks indicative of shared influences were the most common feature in correlations between pairs of raphé neurons, whereas correlated raphé-PRG and raphé-VRC neuron pairs displayed predominantly offset peaks and troughs, features suggesting a paucisynaptic influence of one neuron on the other. Overall, offset correlogram features provided evidence for 33 VRC-to-raphé-to-PRG and 45 PRG-to-raphé-to-VRC correlational linkage chains with one or two intermediate raphé neurons. The results support a respiratory network architecture with parallel VRC-to-PRG and PRG-to-VRC links operating through intervening midline circuits, and suggest that raphé neurons contribute to the respiratory modulation of PRG neurons and shape the respiratory motor pattern through coordinated divergent actions on both the PRG and VRC.

Functional connectivity in the pontomedullary respiratory network.

Current models propose that a neuronal network in the ventrolateral medulla generates the basic respiratory rhythm and that this ventrolateral respiratory column (VRC) is profoundly influenced by the neurons of the pontine respiratory group (PRG). However, functional connectivity among PRG and VRC neurons is poorly understood. This study addressed four model-based hypotheses: 1) the respiratory modulation of PRG neuron populations reflects paucisynaptic actions of multiple VRC populations; 2) functional connections among PRG neurons shape and coordinate their respiratory-modulated activities; 3) the PRG acts on multiple VRC populations, contributing to phase-switching; and 4) neurons with no respiratory modulation located in close proximity to the VRC and PRG have widely distributed actions on respiratory-modulated cells. Two arrays of microelectrodes with individual depth adjustment were used to record sets of spike trains from a total of 145 PRG and 282 VRC neurons in 10 decerebrate, vagotomized, neuromuscularly blocked, ventilated cats. Data were evaluated for respiratory modulation with respect to efferent phrenic motoneuron activity and short-timescale correlations indicative of paucisynaptic functional connectivity using cross-correlation analysis and the "gravity" method. Correlogram features were found for 109 (3%) of the 3,218 pairs composed of a PRG and a VRC neuron, 126 (12%) of the 1,043 PRG-PRG pairs, and 319 (7%) of the 4,340 VRC-VRC neuron pairs evaluated. Correlation linkage maps generated for the data support our four motivating hypotheses and suggest network mechanisms for proposed modulatory functions of the PRG.