Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 µm into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 µm of the murine spinal cord, in vivo.

Neurological and Inflammatory Effects of Radio Frequency and Cryoablation in a Rat Sciatic Nerve Model of Submucosal Nerve Ablation.

BACKGROUND: Minimally-invasive ablation with radio frequency (RF) and cryoablation have been widely adopted to treat conditions with aberrant neural activity such as excessive mucus production in rhinitis, but neurological and inflammatory effects on treated tissues are poorly understood. OBJECTIVE: To gain an understanding of the physiological changes caused by nerve ablation using RF and cryoablation devices. METHODS: Using clinical devices for rhinitis treatment that ablate nerves with access from the nasal cavity, we applied temperature-controlled RF and cryoablation to rat sciatic nerves. To model the ablation through mucosal tissue similarly to the rhinitis procedure, RF ablation and cryoablation were applied through a layer of muscle. RESULTS: Both ablation techniques induced acute and sustained neurodegeneration visualized with histological sections at two days and one month after treatment. After both treatments, rats showed a change in muscle tone, but small increases in sensitivity measured by a von Frey test were only observed 2 days after cryoablation and one month after the RF ablation. Both treatments caused reductions in nerve conduction velocity at one month after treatment. Inflammation in treated nerves and surrounding tissues that persisted to one month. CONCLUSIONS: The two neurolytic devices used in the clinic work similarly by axonal disintegration and which leads to disruption of electrical signals. The data suggest that these methods are effective methods of nerve ablation that could be used to treat diseases related to elevated neuron activity such as rhinitis.

Changes in excitability and ion channel expression in neurons of the major pelvic ganglion in female type II diabetic mice.

Bladder cystopathy and autonomic dysfunction are common complications of diabetes, and have been associated with changes in ganglionic transmission and some measures of neuronal excitability in male mice. To determine whether type II diabetes also impacts excitability of ganglionic neurons in females, we investigated neuronal excitability and firing properties, as well as underlying ion channel expression, in major pelvic ganglion (MPG) neurons in control, 10-week, and 21-week Leprdb/db mice. Type II diabetes in Leprdb/db animals caused a non-linear change in excitability and firing properties of MPG neurons. At 10 weeks, cells exhibited increased excitability as demonstrated by an increased likelihood of firing multiple spikes upon depolarization, decreased rebound spike latency, and overall narrower action potential half-widths as a result of increased depolarization and repolarization slopes. Conversely, at 21 weeks MPG neurons of Leprdb/db mice reversed these changes, with spiking patterns and action-potential properties largely returning to control levels. These changes are associated with numerous time-specific changes in calcium, sodium, and potassium channel subunit mRNA levels. However, Principal Components Analysis of channel expression patterns revealed that rectification of excitability is not simply a return to control levels, but rather a distinct ion channel expression profile in 21-week Leprdb/db neurons. These data indicate that type II diabetes can impact the excitability of post-ganglionic, autonomic neurons of female mice, and suggest that the non-linear progression of these properties with diabetes may be the result of compensatory changes in channel expression that act to rectify disrupted firing patterns of Leprdb/db MPG neurons.

Surgical preparations, labeling strategies, and optical techniques for cell-resolved, in vivo imaging in the mouse spinal cord.

In vivo optical imaging has enabled detailed studies of cellular dynamics in the brain of rodents in both healthy and diseased states. Such studies were made possible by three advances: surgical preparations that give optical access to the brain; strategies for in vivo labeling of cells with structural and functional fluorescent indicators; and optical imaging techniques that are relatively insensitive to light scattering by tissue. In vivo imaging in the rodent spinal cord has lagged behind than that in the brain, largely due to the anatomy around the spinal cord that complicates the surgical preparation, and to the strong optical scattering of the dorsal white matter that limits the ability to image deep into the spinal cord. Here, we review recent advances in surgical methods, labeling strategies, and optical tools that have enabled in vivo, high-resolution imaging of the dynamic behaviors of cells in the spinal cord in mice. Surgical preparations that enable long-term optical access and robust stabilization of the spinal cord are now available. Labeling strategies that have been used in the spinal cord tend to follow those that have been used in the brain, and some recent advances in genetically-encoded labeling strategies remain to be capitalized on. The optical imaging methods used to date, including two photon excited fluorescence microscopy, are largely limited to imaging the superficial layers of the spinal cord by the optical scattering of the white matter. Finally, we show preliminary data that points to the use of higher-order nonlinear optical processes, such as three photon excited fluorescence, as a means to image deeper into the mouse spinal cord.

Removal of endogenous neuromodulators in a small motor network enhances responsiveness to neuromodulation.

We studied the changes in sensitivity to a peptide modulator, crustacean cardioactive peptide (CCAP), as a response to loss of endogenous modulation in the stomatogastric ganglion (STG) of the crab Cancer borealis Our data demonstrate that removal of endogenous modulation for 24 h increases the response of the lateral pyloric (LP) neuron of the STG to exogenously applied CCAP. Increased responsiveness is accompanied by increases in CCAP receptor (CCAPr) mRNA levels in LP neurons, requires de novo protein synthesis, and can be prevented by coincubation for the 24-h period with exogenous CCAP. These results suggest that there is a direct feedback from loss of CCAP signaling to the production of CCAPr that increases subsequent response to the ligand. However, we also demonstrate that the modulator-evoked membrane current (IMI) activated by CCAP is greater in magnitude after combined loss of endogenous modulation and activity compared with removal of just hormonal modulation. These results suggest that both receptor expression and an increase in the target conductance of the CCAP G protein-coupled receptor are involved in the increased response to exogenous hormone exposure following experimental loss of modulation in the STG.NEW & NOTEWORTHY The nervous system shows a tremendous amount of plasticity. More recently there has been an appreciation for compensatory actions that stabilize output in the face of perturbations to normal activity. In this study we demonstrate that neurons of the crustacean stomatogastric ganglion generate apparent compensatory responses to loss of peptide neuromodulation, adding to the repertoire of mechanisms by which the stomatogastric nervous system can regulate and stabilize its own output.

Deep sequencing of transcriptomes from the nervous systems of two decapod crustaceans to characterize genes important for neural circuit function and modulation.

BACKGROUND: Crustaceans have been studied extensively as model systems for nervous system function from single neuron properties to behavior. However, lack of molecular sequence information and tools have slowed the adoption of these physiological systems as molecular model systems. In this study, we sequenced and performed de novo assembly for the nervous system transcriptomes of two decapod crustaceans: the Jonah crab (Cancer borealis) and the American lobster (Homarus americanus). RESULTS: Forty-two thousand, seven hundred sixty-six and sixty thousand, two hundred seventy-three contigs were assembled from C. borealis and H. americanus respectively, representing 9,489 and 11,061 unique coding sequences. From these transcripts, genes associated with neural function were identified and manually curated to produce a characterization of multiple gene families important for nervous system function. This included genes for 34 distinct ion channel types, 17 biogenic amine and 5 GABA receptors, 28 major transmitter receptor subtypes including glutamate and acetylcholine receptors, and 6 gap junction proteins - the Innexins. CONCLUSION: With this resource, crustacean model systems are better poised for incorporation of modern genomic and molecular biology technologies to further enhance the interrogation of fundamentals of nervous system function.

Electrical coupling and innexin expression in the stomatogastric ganglion of the crab Cancer borealis.

Gap junctions are intercellular channels that allow for the movement of small molecules and ions between the cytoplasm of adjacent cells and form electrical synapses between neurons. In invertebrates, the gap junction proteins are coded for by the innexin family of genes. The stomatogastric ganglion (STG) in the crab Cancer borealis contains a small number of identified and electrically coupled neurons. We identified Innexin 1 (Inx1), Innexin 2 (Inx2), Innexin 3 (Inx3), Innexin 4 (Inx4), Innexin 5 (Inx5), and Innexin 6 (Inx6) members of the C. borealis innexin family. We also identified six members of the innexin family from the lobster Homarus americanus transcriptome. These innexins show significant sequence similarity to other arthropod innexins. Using in situ hybridization and reverse transcriptase-quantitative PCR (RT-qPCR), we determined that all the cells in the crab STG express multiple innexin genes. Electrophysiological recordings of coupling coefficients between identified pairs of pyloric dilator (PD) cells and PD-lateral posterior gastric (LPG) neurons show that the PD-PD electrical synapse is nonrectifying while the PD-LPG synapse is apparently strongly rectifying.

Activity-dependent feedback regulates correlated ion channel mRNA levels in single identified motor neurons.

Neurons generate cell-specific outputs via interactions of conductances carried by ion channel proteins that are homeostatically regulated to maintain key quantitative relationships among subsets of conductances. Given the challenges of both normal channel protein turnover and short-term plasticity, how is the balance of membrane conductances maintained over long-term timescales to ensure stable electrophysiological phenotype? One possible mechanism is to dynamically regulate production of channel protein via feedback that constrains relationships at the channel mRNA level. Recent modeling work has postulated that such mRNA relationships could emerge as a result of activity-dependent homeostatic tuning rules that ensure an appropriate ratio of mRNA for key ion channels is maintained to preserve robust cellular output. Yet, this has never been demonstrated in biological neurons. In this study, we quantified multiple ion channel mRNAs from single identified motor neurons of the stomatogastric ganglion to determine whether correlations among channel mRNAs are actively maintained, and, if so, by what form of feedback. In these neurons, we identified correlations among mRNAs for voltage-gated calcium and potassium channels. By performing experiments that decoupled activity, synaptic connectivity, and neuromodulatory state, we determined that correlated channel mRNAs are maintained by an activity-dependent process. This is the first study to demonstrate that distinct relationships across channel mRNAs are dynamically maintained in an activity-dependent manner. This feedback from cellular activity to coordinated transcriptome-level interactions represents a novel aspect of regulation of neuronal output with implications for long-term stability of neuron function.

Dietary fish oil substitution alters the eicosanoid profile in ankle joints of mice during Lyme infection.

Dietary ingestion of (n-3) PUFA alters the production of eicosanoids and can suppress chronic inflammatory and autoimmune diseases. The extent of changes in eicosanoid production during an infection of mice fed a diet high in (n-3) PUFA, however, has not, to our knowledge, been reported. We fed mice a diet containing either 18% by weight soybean oil (SO) or a mixture with fish oil (FO), FO:SO (4:1 ratio), for 2 wk and then infected them with Borrelia burgdorferi. We used an MS-based lipidomics approach and quantified changes in eicosanoid production during Lyme arthritis development over 21 d. B. burgdorferi infection induced a robust production of prostanoids, mono-hydroxylated metabolites, and epoxide-containing metabolites, with 103 eicosanoids detected of the 139 monitored. In addition to temporal and compositional changes in the eicosanoid profile, dietary FO substitution increased the accumulation of 15-deoxy PGJ(2), an antiinflammatory metabolite derived from arachidonic acid. Chiral analysis of the mono-hydroxylated metabolites revealed they were generated from primarily nonenzymatic mechanisms. Although dietary FO substitution reduced the production of inflammatory (n-6) fatty acid-derived eicosanoids, no change in the host inflammatory response or development of disease was detected.

Restoration of descending inputs fails to rescue activity following deafferentation of a motor network.

Motor networks such as the pyloric network of the stomatogastric ganglion often require descending neuromodulatory inputs to initiate, regulate, and modulate their activity and their synaptic connectivity to manifest physiologically appropriate output. Prolonged removal of these descending inputs often results in a compensatory response that alters the inputs themselves, their targets, or both. Using the pyloric network of the crab, Cancer borealis, we investigated whether isolation of motor networks would result in alterations that change the responses of these networks to restored modulatory input. We used a reversible block with isotonic sucrose to transiently alter descending inputs into the pyloric network of the crab stomatogastric ganglion. Using this method, we found that blocking neuromodulatory inputs caused a reduced ability for subsequently restored modulatory projections to appropriately generate network output. Our results suggest that this could be due to changes in activity of descending projection neurons as well as changes in sensitivity to neuromodulators of the target neurons that develop over the time course of the blockade. These findings suggest that although homeostatic plasticity may play a critical role in recovery of functional output in a deafferented motor network, the results of these compensatory changes may alter the network such that restored inputs no longer function appropriately.