Recent GPN Publications

The inhibitory thermal effects of focused ultrasound on an identified, single motoneuron

April 15, 2021 - 5:00am

eNeuro. 2021 Apr 13:ENEURO.0514-20.2021. doi: 10.1523/ENEURO.0514-20.2021. Online ahead of print.


Focused ultrasound (US) is an emerging neuromodulation technology that has gained much attention due to its ability to modulate, noninvasively, neuronal activity in a variety of animals, including humans. However, there has been considerable debate about exactly which types of neurons can be influenced and what underlying mechanisms are in play. Are US-evoked motor changes driven indirectly by activated mechanosensory inputs, or more directly via central interneurons or motoneurons? Although it has been shown that US can mechanically depolarize mechanosensory neurons, there are no studies that have yet tested how identified motoneurons respond directly to US and what the underlying mechanism might be. Here, we examined the effects of US on a single, identified motoneuron within a well-studied and tractable invertebrate preparation, the medicinal leech, Hirudo verbana Our approach aimed to clarify single neuronal responses to US, which may be obscured in other studies whereby US is applied across a diverse population of cells. We found that US has the ability to inhibit tonic spiking activity through a predominately thermal mechanism. US-evoked effects persisted after blocking synaptic inputs, indicating that its actions were direct. Experiments also revealed that US-comparable heating blocked the axonal conduction of spontaneous action potentials. Finally, we found no evidence that US had significant mechanical effects on the neurons tested, a finding counter to prevailing views. We conclude that a non-sensory neuron can be directly inhibited via a thermal mechanism, a finding that holds promise for clinical neuromodulatory applications.SIGNIFICANCE STATEMENTMuch of the enthusiasm regarding focused ultrasound (US) neuromodulation stems from human and other mammalian noninvasive transcranial stimulation and its effects on evoked potentials or motor activity. However, there is considerable debate in the field of US neuromodulation about exactly which types of neurons can be influenced, what the direct outcomes are, and what underlying mechanisms are responsible. In our study, conducted in the medicinal leech Hirudo verbana, we examine for the first time whether a motoneuron could respond to US, which was accomplished at the single-cell level. We found that under conditions whereby US generated sufficient heat (2-3°C), an inhibitory response was generated. These results have important implications for the noninvasive treatment of chronic pain and other neural disorders.

PMID:33853851 | DOI:10.1523/ENEURO.0514-20.2021

Differential impact of inhibitory G protein signaling pathways in ventral tegmental area dopamine neurons on behavioral sensitivity to cocaine and morphine

March 12, 2021 - 5:00am

eNeuro. 2021 Mar 10:ENEURO.0081-21.2021. doi: 10.1523/ENEURO.0081-21.2021. Online ahead of print.


Drugs of abuse engage overlapping but distinct molecular and cellular mechanisms to enhance dopamine (DA) signaling in the mesocorticolimbic circuitry. DA neurons of the ventral tegmental area (VTA) are key substrates of drugs of abuse and have been implicated in addiction-related behaviors. Enhanced VTA DA neurotransmission evoked by drugs of abuse can engage inhibitory G protein-dependent feedback pathways, mediated by GABAB receptors (GABABRs) and D2 DA receptors (D2Rs). Chemogenetic inhibition of VTA DA neurons potently suppressed baseline motor activity, as well as the motor-stimulatory effect of cocaine and morphine, confirming the critical influence of VTA DA neurons and inhibitory G protein signaling in these neurons on this addiction-related behavior. To resolve the relative influence of GABABR- and D2R-dependent signaling pathways in VTA DA neurons on behavioral sensitivity to drugs of abuse, we developed a neuron-specific viral CRISPR/Cas9 approach to ablate D2R and GABABR in VTA DA neurons. Ablation of GABABR or D2R did not impact baseline physiological properties or excitability of VTA DA neurons, but it did preclude the direct somatodendritic inhibitory influence of GABABR or D2R activation. D2R ablation potentiated the motor-stimulatory effect of cocaine in male and female mice, whereas GABABR ablation selectively potentiated cocaine-induced activity in male subjects only. Neither D2R nor GABABR ablation impacted morphine-induced motor activity. Collectively, our data show that cocaine and morphine differ in the extent to which they engage inhibitory G protein-dependent feedback pathways in VTA DA neurons and highlight key sex differences that may impact susceptibility to various facets of addiction.SIGNIFICANCE STATEMENTAlthough inhibitory G protein-dependent signaling involving the GABAB (GABABR) and D2 dopamine receptor (D2R) in VTA DA neurons is thought to limit DA neurotransmission evoked by drugs of abuse, their relative impact on behavioral sensitivity to such drugs is unclear. Using a neuron-specific viral CRISPR/Cas9 approach, we show that that loss of D2R in VTA DA neurons enhances behavioral sensitivity to systemic administration of cocaine in male and female mice, whereas loss of GABABR enhances cocaine sensitivity only in males. Neither GABABR nor D2R ablation impacted behavioral sensitivity to morphine. Thus, differential engagement of inhibitory feedback pathways in VTA DA neurons likely contributes to drug-specific neurophysiological and behavioral effects and may underlie sex differences associated with some facets of addiction.

PMID:33707203 | DOI:10.1523/ENEURO.0081-21.2021

Lung Mediated Auditory Contrast Enhancement Improves the Signal-to-Noise Ratio for Communication in Frogs

March 5, 2021 - 5:00am

Curr Biol. 2021 Feb 23:S0960-9822(21)00113-5. doi: 10.1016/j.cub.2021.01.048. Online ahead of print.


Environmental noise is a major source of selection on animal sensory and communication systems. The acoustic signals of other animals represent particularly potent sources of noise for chorusing insects, frogs, and birds, which contend with a multi-species analog of the human "cocktail party problem" (i.e., our difficulty following speech in crowds). However, current knowledge of the diverse adaptations that function to solve noise problems in nonhuman animals remains limited. Here, we show that a lung-to-ear sound transmission pathway in frogs serves a heretofore unknown noise-control function in vertebrate hearing and sound communication. Inflated lungs improve the signal-to-noise ratio for communication by enhancing the spectral contrast in received vocalizations in ways analogous to signal processing algorithms used in hearing aids and cochlear implants. Laser vibrometry revealed that the resonance of inflated lungs selectively reduces the tympanum's sensitivity to frequencies between the two spectral peaks present in conspecific mating calls. Social network analysis of continent-scale citizen science data on frog calling behavior revealed that the calls of other frog species in multi-species choruses can be a prominent source of environmental noise attenuated by the lungs. Physiological modeling of peripheral frequency tuning indicated that inflated lungs could reduce both auditory masking and suppression of neural responses to mating calls by environmental noise. Together, these data suggest an ancient adaptation for detecting sound via the lungs has been evolutionarily co-opted to create auditory contrast enhancement that contributes to solving a multi-species cocktail party problem.

PMID:33667371 | DOI:10.1016/j.cub.2021.01.048

Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

February 24, 2021 - 5:00am

Sci Rep. 2021 Feb 23;11(1):4367. doi: 10.1038/s41598-021-83666-3.


Protein prenylation involves the attachment of one or two isoprenoid group(s) onto cysteine residues positioned near the C-terminus. This modification is essential for many signal transduction processes. In this work, the use of the probe C15AlkOPP for metabolic labeling and identification of prenylated proteins in a variety of cell lines and primary cells is explored. Using a single isoprenoid analogue, 78 prenylated protein groups from the three classes of prenylation substrates were identified including three novel prenylation substrates in a single experiment. Applying this method to three brain-related cell lines including neurons, microglia, and astrocytes showed substantial overlap (25%) in the prenylated proteins identified. In addition, some unique prenylated proteins were identified in each type. Eight proteins were observed exclusively in neurons, five were observed exclusively in astrocytes and three were observed exclusively in microglia, suggesting their unique roles in these cells. Furthermore, inhibition of farnesylation in primary astrocytes revealed the differential responses of farnesylated proteins to an FTI. Importantly, these results provide a list of 19 prenylated proteins common to all the cell lines studied here that can be monitored using the C15AlkOPP probe as well as a number of proteins that were observed in only certain cell lines. Taken together, these results suggest that this chemical proteomic approach should be useful in monitoring the levels and exploring the underlying role(s) of prenylated proteins in various diseases.

PMID:33623102 | DOI:10.1038/s41598-021-83666-3

Through the looking glass: a review of cranial window technology for optical access to the brain

February 18, 2021 - 5:00am

J Neurosci Methods. 2021 Feb 15:109100. doi: 10.1016/j.jneumeth.2021.109100. Online ahead of print.


Deciphering neurologic function is a daunting task, requiring understanding the neuronal networks and emergent properties that arise from the interactions among single neurons. Mechanistic insights into neuronal networks require tools that simultaneously assess both single neuron activity and the consequent mesoscale output. The development of cranial window technologies, in which the skull is thinned or replaced with a synthetic optical interface, has enabled monitoring neuronal activity from subcellular to mesoscale resolution in awake, behaving animals when coupled with advanced microscopy techniques. Here we review recent achievements in cranial window technologies, appraise the relative merits of each design and discuss the future research in cranial window design.

PMID:33600850 | DOI:10.1016/j.jneumeth.2021.109100

Continuing the Dialog on Human-Animal Chimerism: Response to Bolo, Wills, and Maschke

February 10, 2021 - 5:00am

Stem Cell Reports. 2021 Feb 9;16(2):227. doi: 10.1016/j.stemcr.2021.01.003.


PMID:33567292 | DOI:10.1016/j.stemcr.2021.01.003

Function of Renal Nerves in Kidney Physiology and Pathophysiology

February 10, 2021 - 5:00am

Annu Rev Physiol. 2021 Feb 10;83:429-450. doi: 10.1146/annurev-physiol-031620-091656.


Renal sympathetic (efferent) nerves play an important role in the regulation of renal function, including glomerular filtration, sodium reabsorption, and renin release. The kidney is also innervated by sensory (afferent) nerves that relay information to the brain to modulate sympathetic outflow. Hypertension and other cardiometabolic diseases are linked to overactivity of renal sympathetic and sensory nerves, but our mechanistic understanding of these relationships is limited. Clinical trials of catheter-based renal nerve ablation to treat hypertension have yielded promising results. Therefore, a greater understanding of how renal nerves control the kidney under physiological and pathophysiological conditions is needed. In this review, we provide an overview of the current knowledge of the anatomy of efferent and afferent renal nerves and their functions in normal and pathophysiological conditions. We also suggest further avenues of research for development of novel therapies targeting the renal nerves.

PMID:33566672 | DOI:10.1146/annurev-physiol-031620-091656

Moderately elevated glucocorticoids increase mate choosiness but do not affect sexual proceptivity or preferences in female gray treefrogs

February 8, 2021 - 5:00am

Horm Behav. 2021 Feb 5:104950. doi: 10.1016/j.yhbeh.2021.104950. Online ahead of print.


Glucocorticoids (GCs) are rarely studied in the context of female mate choice, despite the expression of receptors for these products in sexual, sensory and decision-making brain areas. Here we investigated the effects of GC concentrations on three aspects of female sexual behavior in breeding Cope's gray treefrogs (Hyla chrysoscelis): proceptivity-a measure of sexual motivation, intraspecific mate preferences, and mate choosiness. To our knowledge this is the first experimental study on the endocrine basis of mate choosiness. We predicted that mate choosiness-forfeiting an initial mate preference to pursue a suddenly more attractive mate-would be particularly impacted by elevated GCs with moderate GC levels associated with greater choosiness. We found support for this predicted inverted-U relationship. Females in the control group (no injection) showed no change in choosiness across timepoints. In contrast, females in the vehicle, Low (20 ng g-1) and High (180 ng g-1) corticosterone groups exhibited a nominal decline in choosiness after injection, suggesting that the experience of injection has little or perhaps slightly suppressive effects on female choosiness. Females in the moderate dose group (60 ng g-1), however, exhibited a significant increase (>100%) in choosiness. Further, we found no effect of elevated GCs on sexual proceptivity or the species-typical preference for longer calls. These findings may reflect a buffering of primary sensory areas in the brain against elevated GCs. The recruitment of other cognitive processes during active decision-making, however, may facilitate GC modulation of mate choosiness, thereby promoting tactical plasticity at this critical life history juncture.

PMID:33556376 | DOI:10.1016/j.yhbeh.2021.104950

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

February 8, 2021 - 5:00am

J Vis Exp. 2021 Jan 21;(167). doi: 10.3791/61599.


A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

PMID:33554975 | DOI:10.3791/61599