MEMRI for visualizing brain activity after auditory stimulation in frogs.

Anuran amphibians are common model organisms in bioacoustics and neurobiology. To date, however, most available methods for studying auditory processing in frogs are highly invasive and thus do not allow for longitudinal study designs, nor do they provide a global view of the brain, which substantially limits the questions that can be addressed. The goal of this study was to identify areas in the frog brain that are responsible for auditory processing using in vivo manganese-enhanced MRI (MEMRI). We were interested in determining if the neural processing of socially relevant acoustic stimuli (e.g., species-specific calls) engages a specific pattern of brain activation that differs from patterns elicited by less- or nonrelevant acoustic signals. We thus designed an experiment, in which we presented three different types of acoustic stimuli (species-specific calls, band-limited noise, or silence) to fully awake northern leopard frogs (Rana pipiens) and then conducted MEMRI T1-weighted imaging to investigate differences in signal intensity due to manganese uptake as an indication of brain activity across all three conditions. We found the greatest change in signal intensity within the torus semicircularis (the principal central auditory region), the habenula, and the paraphysis of frogs that had been exposed to conspecific calls compared with noise or silence conditions. Stimulation with noise did not result in the same activation patterns, indicating that signals with contrasting social relevance are differentially processed in these areas of the amphibian brain. MEMRI provides a powerful approach to studying brain activity with high spatial resolution in frogs. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Optogenetic stimulation of adrenergic C1 neurons causes sleep state-dependent cardiorespiratory stimulation and arousal with sighs in rats.

RATIONALE: The rostral ventrolateral medulla (RVLM) contains central respiratory chemoreceptors (retrotrapezoid nucleus, RTN) and the sympathoexcitatory, hypoxia-responsive C1 neurons. Simultaneous optogenetic stimulation of these neurons produces vigorous cardiorespiratory stimulation, sighing, and arousal from non-REM sleep. OBJECTIVES: To identify the effects that result from selectively stimulating C1 cells. METHODS: A Cre-dependent vector expressing channelrhodopsin 2 (ChR2) fused with enhanced yellow fluorescent protein or mCherry was injected into the RVLM of tyrosine hydroxylase (TH)-Cre rats. The response of ChR2-transduced neurons to light was examined in anesthetized rats. ChR2-transduced C1 neurons were photoactivated in conscious rats while EEG, neck muscle EMG, blood pressure (BP), and breathing were recorded. MEASUREMENTS AND MAIN RESULTS: Most ChR2-expressing neurons (95%) contained C1 neuron markers and innervated the spinal cord. RTN neurons were not transduced. While the rats were under anesthesia, the C1 cells were faithfully activated by each light pulse up to 40 Hz. During quiet resting and non-REM sleep, C1 cell stimulation (20 s, 2-20 Hz) increased BP and respiratory frequency and produced sighs and arousal from non-REM sleep. Arousal was frequency-dependent (85% probability at 20 Hz). Stimulation during REM sleep increased BP, but had no effect on EEG or breathing. C1 cell-mediated breathing stimulation was occluded by hypoxia (12% FIO2), but was unchanged by 6% FiCO2. CONCLUSIONS: C1 cell stimulation reproduces most effects of acute hypoxia, specifically cardiorespiratory stimulation, sighs, and arousal. C1 cell activation likely contributes to the sleep disruption and adverse autonomic consequences of sleep apnea. During hypoxia (awake) or REM sleep, C1 cell stimulation increases BP but no longer stimulates breathing.

The orexinergic neurons receive synaptic input from C1 cells in rats.

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N-methyl transferase-immunoreactive (PNMT-ir) axons were detected among orexin-ir somata, and close appositions between PNMT-ir axonal varicosities and orexin-ir profiles were observed. The existence of synapses between PNMT-ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2-mCherry under the control of the catecholaminergic neuron-selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre-dependent adeno-associated vector and TH-Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT-ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin-ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense-core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1-orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep.

Analysis of protein-RNA complexes involving a RNA recognition motif engineered to bind hairpins with seven- and eight-nucleotide loops.

U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.

Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.

C1 catecholaminergic neurons and neurons of the retrotrapezoid nucleus are integrative nodes within the brain stem network regulating cardiorespiratory reflexes elicited by hypoxia and hypercapnia, stimuli that also produce arousal from sleep. In the present study, Channelrhodopsin-2 was selectively introduced into these neurons with a lentiviral vector to determine whether their selective activation also produces arousal in sleeping rats. Sleep stages were identified from electroencephalographic and neck muscle electromyographic recordings. Breathing was measured using unrestrained whole body plethysmography and blood pressure by telemetry. During nonrapid eye movement sleep, unilateral photostimulation of the C1 region caused arousal in 83.0±14.7% of trials and immediate and intense cardiorespiratory activation. Arousal during photostimulation was also observed during rapid eye movement sleep (41.9±5.6% of trials), but less reliably than during nonrapid eye movement sleep. The cardiorespiratory responses elicited by photostimulation were dramatically smaller during rapid eye movement sleep than nonrapid eye movement sleep or wakefulness. Systemic α1-adrenoreceptor blockade reduced the cardiorespiratory effects of photostimulation but had no effect on the arousal caused by photostimulation during nonrapid eye movement sleep. Postmortem histology showed that neurons expressing Channelrhodopsin 2-mCherry were predominantly catecholaminergic (81%). These results show that selective activation of C1 and retrotrapezoid nucleus neurons produces state-dependent arousal and cardiorespiratory stimulation. These neurons, which are powerfully activated by chemoreceptor stimulation, may contribute to the sleep disruption associated with obstructive sleep apnea.

Selective optogenetic activation of rostral ventrolateral medullary catecholaminergic neurons produces cardiorespiratory stimulation in conscious mice.

Activation of rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons e.g., by hypoxia is thought to increase sympathetic outflow thereby raising blood pressure (BP). Here we test whether these neurons also regulate breathing and cardiovascular variables other than BP. Selective expression of ChR2-mCherry by RVLM-CA neurons was achieved by injecting Cre-dependent vector AAV2-EF1α-DIO-ChR2-mCherry unilaterally into the brainstem of dopamine-ß-hydroxylase(Cre/0) mice. Photostimulation of RVLM-CA neurons increased breathing in anesthetized and conscious mice. In conscious mice, photostimulation primarily increased breathing frequency and this effect was fully occluded by hypoxia (10% O(2)). In contrast, the effects of photostimulation were largely unaffected by hypercapnia (3 and 6% CO(2)). The associated cardiovascular effects were complex (slight bradycardia and hypotension) and, using selective autonomic blockers, could be explained by coactivation of the sympathetic and cardiovagal outflows. ChR2-positive RVLM-CA neurons expressed VGLUT2 and their projections were mapped. Their complex cardiorespiratory effects are presumably mediated by their extensive projections to supraspinal sites such as the ventrolateral medulla, the dorsal vagal complex, the dorsolateral pons, and selected hypothalamic nuclei (dorsomedial, lateral, and paraventricular nuclei). In sum, selective optogenetic activation of RVLM-CA neurons in conscious mice revealed two important novel functions of these neurons, namely breathing stimulation and cardiovagal outflow control, effects that are attenuated or absent under anesthesia and are presumably mediated by the numerous supraspinal projections of these neurons. The results also suggest that RVLM-CA neurons may underlie some of the acute respiratory response elicited by carotid body stimulation but contribute little to the central respiratory chemoreflex.

Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

The C1 neurons are a nodal point for blood pressure control and other autonomic responses. Here we test whether these rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus (DMV). After injecting Cre-dependent adeno-associated virus (AAV2) DIO-Ef1α-channelrhodopsin2(ChR2)-mCherry (AAV2) into the RVLM of dopamine-ß-hydroxylase Cre transgenic mice (DßH(Cre/0)), mCherry was detected exclusively in RVLM-CA neurons. Within the DMV >95% mCherry-immunoreactive(ir) axonal varicosities were tyrosine hydroxylase (TH)-ir and the same proportion were vesicular glutamate transporter 2 (VGLUT2)-ir. VGLUT2-mCherry colocalization was virtually absent when AAV2 was injected into the RVLM of DßH(Cre/0);VGLUT2(flox/flox) mice, into the caudal VLM (A1 noradrenergic neuron-rich region) of DßH(Cre/0) mice or into the raphe of ePet(Cre/0) mice. Following injection of AAV2 into RVLM of TH-Cre rats, phenylethanolamine N-methyl transferase and VGLUT2 immunoreactivities were highly colocalized in DMV within EYFP-positive or EYFP-negative axonal varicosities. Ultrastructurally, mCherry terminals from RVLM-CA neurons in DßH(Cre/0) mice made predominantly asymmetric synapses with choline acetyl-transferase-ir DMV neurons. Photostimulation of ChR2-positive axons in DßH(Cre/0) mouse brain slices produced EPSCs in 71% of tested DMV preganglionic neurons (PGNs) but no IPSCs. Photostimulation (20 Hz) activated PGNs up to 8 spikes/s (current-clamp). EPSCs were eliminated by tetrodotoxin, reinstated by 4-aminopyridine, and blocked by ionotropic glutamate receptor blockers. In conclusion, VGLUT2 is expressed by RVLM-CA (C1) neurons in rats and mice regardless of the presence of AAV2, the C1 neurons activate DMV parasympathetic PGNs monosynaptically and this connection uses glutamate as an ionotropic transmitter.

Cholinergic neurons in the mouse rostral ventrolateral medulla target sensory afferent areas.

The rostral ventrolateral medulla (RVLM) primarily regulates respiration and the autonomic nervous system. Its medial portion (mRVLM) contains many choline acetyltransferase (ChAT)-immunoreactive (ir) neurons of unknown function. We sought to clarify the role of these cholinergic cells by tracing their axonal projections. We first established that these neurons are neither parasympathetic preganglionic neurons nor motor neurons because they did not accumulate intraperitoneally administered Fluorogold. We traced their axonal projections by injecting a Cre-dependent vector (floxed-AAV2) expressing either GFP or mCherrry into the mRVLM of ChAT-Cre mice. Transduced neurons expressing GFP or mCherry were confined to the injection site and were exclusively ChAT-ir. Their axonal projections included the dorsal column nuclei, medullary trigeminal complex, cochlear nuclei, superior olivary complex and spinal cord lamina III. For control experiments, the floxed-AAV2 (mCherry) was injected into the RVLM of dopamine beta-hydroxylase-Cre mice. In these mice, mCherry was exclusively expressed by RVLM catecholaminergic neurons. Consistent with data from rats, these catecholaminergic neurons targeted brain regions involved in autonomic and endocrine regulation. These regions were almost totally different from those innervated by the intermingled mRVLM-ChAT neurons. This study emphasizes the advantages of using Cre-driver mouse strains in combination with floxed-AAV2 to trace the axonal projections of chemically defined neuronal groups. Using this technique, we revealed previously unknown projections of mRVLM-ChAT neurons and showed that despite their close proximity to the cardiorespiratory region of the RVLM, these cholinergic neurons regulate sensory afferent information selectively and presumably have little to do with respiration or circulatory control.

Pre-Bötzinger complex receives glutamatergic innervation from galaninergic and other retrotrapezoid nucleus neurons.

The retrotrapezoid nucleus (RTN) contains CO(2) -responsive neurons that regulate breathing frequency and amplitude. These neurons (RTN-Phox2b neurons) contain the transcription factor Phox2b, vesicular glutamate transporter 2 (VGLUT2) mRNA, and a subset contains preprogalanin mRNA. We wished to determine whether the terminals of RTN-Phox2b neurons contain galanin and VGLUT2 proteins, to identify the specific projections of the galaninergic subset, to test whether RTN-Phox2b neurons contact neurons in the pre-Bötzinger complex, and to identify the ultrastructure of these synapses. The axonal projections of RTN-Phox2b neurons were traced by using biotinylated dextran amine (BDA), and many BDA-ir boutons were found to contain galanin immunoreactivity. RTN galaninergic neurons had ipsilateral projections that were identical with those of this nucleus at large: the ventral respiratory column, the caudolateral nucleus of the solitary tract, and the pontine Kölliker-Fuse, intertrigeminal region, and lateral parabrachial nucleus. For ultrastructural studies, RTN-Phox2b neurons (galaninergic and others) were transfected with a lentiviral vector that expresses mCherry almost exclusively in Phox2b-ir neurons. After spinal cord injections of a catecholamine neuron-selective toxin, there was a depletion of C1 neurons in the RTN area; thus it was determined that the mCherry-positive terminals located in the pre-Bötzinger complex originated almost exclusively from the RTN-Phox2b (non-C1) neurons. These terminals were generally VGLUT2-immunoreactive and formed numerous close appositions with neurokinin-1 receptor-ir pre-Bötzinger complex neurons. Their boutons (n = 48) formed asymmetric synapses filled with small clear vesicles. In summary, RTN-Phox2b neurons, including the galaninergic subset, selectively innervate the respiratory pattern generator plus a portion of the dorsolateral pons. RTN-Phox2b neurons establish classic excitatory glutamatergic synapses with pre-Bötzinger complex neurons presumed to generate the respiratory rhythm.

Phox2b-expressing neurons of the parafacial region regulate breathing rate, inspiration, and expiration in conscious rats.

The retrotrapezoid nucleus contains Phox2b-expressing glutamatergic neurons (RTN-Phox2b neurons) that regulate breathing in a CO2-dependent manner. Here we use channelrhodopsin-based optogenetics to explore how these neurons control breathing in conscious and anesthetized adult rats. Respiratory entrainment (pacing) of breathing frequency (fR) was produced over 57% (anesthetized) and 28% (conscious) of the natural frequency range by burst activation of RTN-Phox2b neurons (3-8 × 0.5-20 ms pulses at 20 Hz). In conscious rats, pacing under normocapnic conditions increased tidal volume (V(T)) and each inspiration was preceded by active expiration, denoting abdominal muscle contraction. During long-term pacing V(T) returned to prestimulation levels, suggesting that central chemoreceptors such as RTN-Phox2b neurons regulate V(T) partly independently of their effect on fR. Randomly applied light trains reset the respiratory rhythm and shortened the expiratory phase when the stimulus coincided with late-inspiration or early-expiration. Importantly, continuous (20 Hz) photostimulation of the RTN-Phox2b neurons and a saturating CO2 concentration produced similar effects on breathing that were much larger than those elicited by phasic RTN stimulation. In sum, consistent with their anatomical projections, RTN-Phox2b neurons regulate lung ventilation by controlling breathing frequency, inspiration, and active expiration. Adult RTN-Phox2b neurons can entrain the respiratory rhythm if their discharge is artificially synchronized, but continuous activation of these neurons is much more effective at increasing lung ventilation. These results suggest that RTN-Phox2b neurons are no longer rhythmogenic in adulthood and that their average discharge rate may be far more important than their discharge pattern in driving lung ventilation.

Control of breathing by raphe obscurus serotonergic neurons in mice.

We used optogenetics to determine the global respiratory effects produced by selectively stimulating raphe obscurus (RO) serotonergic neurons in anesthetized mice and to test whether these neurons detect changes in the partial pressure of CO(2), and hence function as central respiratory chemoreceptors. Channelrhodopsin-2 (ChR2) was selectively (∼97%) incorporated into ∼50% of RO serotonergic neurons by injecting AAV2 DIO ChR2-mCherry (adeno-associated viral vector double-floxed inverse open reading frame of ChR2-mCherry) into the RO of ePet-Cre mice. The transfected neurons heavily innervated lower brainstem and spinal cord regions involved in autonomic and somatic motor control plus breathing but eschewed sensory related regions. Pulsed laser photostimulation of ChR2-transfected serotonergic neurons increased respiratory frequency (fR) and diaphragmatic EMG (dEMG) amplitude in relation to the duration and frequency of the light pulses (half saturation, 1 ms; 5-10 Hz). dEMG amplitude and fR increased slowly (half saturation after 10-15 s) and relaxed monoexponentially (tau, 13-15 s). The breathing stimulation was reduced ∼55% by methysergide (broad spectrum serotonin antagonist) and potentiated (∼16%) at elevated levels of inspired CO(2) (8%). RO serotonergic neurons, identified by their entrainment to short light pulses (threshold, 0.1-1 ms) were silent (nine cells) or had a low and regular level of activity (2.1 ± 0.4 Hz; 11 cells) that was not synchronized with respiration. These and nine surrounding neurons with similar characteristics were unaffected by adding up to 10% CO(2) to the breathing mixture. In conclusion, RO serotonergic neurons activate breathing frequency and amplitude and potentiate the central respiratory chemoreflex but do not appear to have a central respiratory chemoreceptor function.

A review of empathy education in nursing.

The ability for nurse educators to improve the empathy skill set of nurses has been the subject of several studies with varied outcomes. The aim of this paper is to review the evidence for empathy education programmes in nursing and make recommendations for future nurse education. A review of CINAHL, Medline, Psych Info and Google Scholar was undertaken using the keywords empathy, person centredness, patient centredness, client centredness, education and nursing. The studies included were required to have measured the effectiveness of empathy training in postgraduate and or undergraduate nurses. The included studies incorporated both qualitative and quantitative methods and were published in peer-reviewed journals. Studies were ranked for level of evidence according to The Joanna Briggs Institute criteria. Seventeen studies from the literature review were found that met the inclusion criteria. Of the 17 studies, 11 reported statistically significant improvements in empathy scores versus six studies that did not. Several variables may affect empathy education that need to be accounted in future studies such as; gender, cultural values and clinical speciality experience. Models of education that show most promise are those that use experiential styles of learning. The studies reviewed demonstrated that it is possible to increase nurses' empathic ability.

Flight performance in night-flying sweat bees suffers at low light levels.

The sweat bee Megalopta (Hymenoptera: Halictidae), unlike most bees, flies in extremely dim light. And although nocturnal insects are often equipped with superposition eyes, which greatly enhance light capture, Megalopta performs visually guided flight with apposition eyes. We examined how light limits Megalopta's flight behavior by measuring flight times and corresponding light levels and comparing them with flight trajectories upon return to the nest. We found the average time to land increased in dim light, an effect due not to slow approaches, but to circuitous approaches. Some landings, however, were quite fast even in the dark. To explain this, we examined the flight trajectories and found that in dim light, landings became increasingly error prone and erratic, consistent with repeated landing attempts. These data agree well with the premise that Megalopta uses visual summation, sacrificing acuity in order to see and fly at the very dimmest light intensities that its visual system allows.

The spectral sensitivity of the lens eyes of a box jellyfish, Tripedalia cystophora (Conant).

Box jellyfish, or cubomedusae (class Cubozoa), are unique among the Cnidaria in possessing lens eyes similar in morphology to those of vertebrates and cephalopods. Although these eyes were described over 100 years ago, there has been no work done on their electrophysiological responses to light. We used an electroretinogram (ERG) technique to measure spectral sensitivity of the lens eyes of the Caribbean species Tripedalia cystophora. The cubomedusae have two kinds of lens eyes, the lower and upper lens eyes. We found that both lens eye types have similar spectral sensitivities, which likely result from the presence of a single receptor type containing a single opsin. The peak sensitivity is to blue-green light. Visual pigment template fits indicate a vitamin A-1 based opsin with peak sensitivity near 500 nm for both eye types.

Advanced optics in a jellyfish eye.

Cubozoans, or box jellyfish, differ from all other cnidarians by an active fish-like behaviour and an elaborate sensory apparatus. Each of the four sides of the animal carries a conspicuous sensory club (the rhopalium), which has evolved into a bizarre cluster of different eyes. Two of the eyes on each rhopalium have long been known to resemble eyes of higher animals, but the function and performance of these eyes have remained unknown. Here we show that box-jellyfish lenses contain a finely tuned refractive index gradient producing nearly aberration-free imaging. This demonstrates that even simple animals have been able to evolve the sophisticated visual optics previously known only from a few advanced bilaterian phyla. However, the position of the retina does not coincide with the sharp image, leading to very wide and complex receptive fields in individual photoreceptors. We argue that this may be useful in eyes serving a single visual task. The findings indicate that tailoring of complex receptive fields might have been one of the original driving forces in the evolution of animal lenses.

Visual ecology and functional morphology of cubozoa (cnidaria).

Jellyfish belong to one of the oldest extant animal phyla, the Cnidaria. The first Cnidaria appear in the fossil record 600 million years ago, preceeding the Cambrian explosion. They are an extremely successful group present in all marine environments and some freshwater environments. In contrast to many animal phyla in which vision is a primary sense Cnidarians do not, generally, employ image forming eyes. One small class stands alone: the Cubozoa. Cubomedusae are commonly known as box jellyfish. They possess image forming eyes (Coates et al., 2001) which certainly evolved independently from other metazoans. Cubomedusae therefore offer a unique perspective on the evolution of image forming eyes. This literature review collects, into one place, what is known about: the multiple eye types of box jellyfish, cubomedusan life history and ecology, and the sensory and neural systems of box jellyfish. Here I discuss how these features set cubomedusae apart from scyphomedusae and hydromedusae. Knowledge in these areas is sparse; the work done to date inspires increased efforts.