A deep learning approach to identifying immunogold particles in electron microscopy images.

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.

An open-source tool for analysis and automatic identification of dendritic spines using machine learning.

Synaptic plasticity, the cellular basis for learning and memory, is mediated by a complex biochemical network of signaling proteins. These proteins are compartmentalized in dendritic spines, the tiny, bulbous, post-synaptic structures found on neuronal dendrites. The ability to screen a high number of molecular targets for their effect on dendritic spine structural plasticity will require a high-throughput imaging system capable of stimulating and monitoring hundreds of dendritic spines in various conditions. For this purpose, we present a program capable of automatically identifying dendritic spines in live, fluorescent tissue. Our software relies on a machine learning approach to minimize any need for parameter tuning from the user. Custom thresholding and binarization functions serve to "clean" fluorescent images, and a neural network is trained using features based on the relative shape of the spine perimeter and its corresponding dendritic backbone. Our algorithm is rapid, flexible, has over 90% accuracy in spine detection, and bundled with our user-friendly, open-source, MATLAB-based software package for spine analysis.

RGS14 Restricts Plasticity in Hippocampal CA2 by Limiting Postsynaptic Calcium Signaling.

Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca2+) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.

Automated Remote Focusing, Drift Correction, and Photostimulation to Evaluate Structural Plasticity in Dendritic Spines.

Long-term structural plasticity of dendritic spines plays a key role in synaptic plasticity, the cellular basis for learning and memory. The biochemical step is mediated by a complex network of signaling proteins in spines. Two-photon imaging techniques combined with two-photon glutamate uncaging allows researchers to induce and quantify structural plasticity in single dendritic spines. However, this method is laborious and slow, making it unsuitable for high throughput screening of factors necessary for structural plasticity. Here we introduce a MATLAB-based module built for Scanimage to automatically track, image, and stimulate multiple dendritic spines. We implemented an electrically tunable lens in combination with a drift correction algorithm to rapidly and continuously track targeted spines and correct sample movements. With a straightforward user interface to design custom multi-position experiments, we were able to adequately image and produce targeted plasticity in multiple dendritic spines using glutamate uncaging. Our methods are inexpensive, open source, and provides up to a five-fold increase in throughput for quantifying structural plasticity of dendritic spines.

The effects of confinement on neuronal growth cone morphology and velocity.

Optimizing growth cone guidance through the use of patterned substrates is important for designing regenerative substrates to aid in recovery from neuronal injury. Using laser ablation, we designed micron-scale patterns capable of confining dissociated mouse cerebellar granule neuron growth cones to channels of different widths ranging from 1.5 to 12 µm. Growth cone dynamics in these channels were observed using time-lapse microscopy. Growth cone area was decreased in channels between 1.5 and 6 µm as compared to that in 12 µm and unpatterned substrates. Growth cone aspect ratio was also affected as narrower channels forced growth cones into a narrow, elongated shape. There was no difference in the overall rate of growth cone advance in uniform channels between 1.5 and 12 µm as compared to growth on unpatterned substrates. The percentage of time growth cones advanced, paused, and retracted was also similar. However, growth cones did respond to changes in confinement: growth cones in narrow lanes rapidly sped up when encountering a wide region and then slowed down as they entered another narrow region. Our results suggest that the rate of neurite extension is not affected by the degree of confinement, but does respond to changes in confinement.

Phasic and tonic fluctuations in brain, muscle, and skin temperatures during motivated drinking behavior in rats: physiological correlates of motivation and reward.

Since brain metabolism is accompanied by heat production, measurement of brain temperature offers a method for assessing global alterations in metabolic neural activity. This approach, high-resolution (5-s bin) temperature recording from the nucleus accumbens (NAcc), temporal muscle, and facial skin, was used to study motivated drinking behavior in rats. Experienced animals were presented with a cup containing 5-ml of Coca-Cola(R) (Coke) beverage that resulted, within certain latencies, in initiation of a continuous chain of licking until all liquid was fully consumed. While cup presentation induced rapid, gradual NAcc temperature increase peaking at the start of drinking, temperatures slowly decreased during Coke consumption, but phasically increased again in the post-consumption period when rats were hyperactive, showing multiple interactions with an empty cup. Muscle temperatures followed a similar pattern, but the changes were weaker and delayed compared to those in the brain. Skin temperature rapidly dropped after cup presentation, steadily maintained at low levels during consumption, and slowly restored during the post-consumption period. Substitution of the expected Coke with either sugar-free Diet Coke(R) or water resulted in numerous drinking attempts but ultimately no consumption. During these tests, locomotor activation was much greater and more prolonged, brain and muscle temperatures increased monophasically, and their elevation was significantly greater than that with regular Coke tests. Food deprivation decreased drinking latencies, did not change the pattern of temperature fluctuations during Coke consumption, but temperature elevations were greater than in controls. Our data suggest sustained neural activation triggered by appetitive stimuli and associated with activational (seeking) aspects of appetitive motivated behavior. This seeking-related activation is rapidly ceased following consumption, suggesting this change as a neural correlate of reward. In contrast, inability to obtain an expected reward maintains neural activation and seeking behavior, resulting in larger deviations in physiological parameters.

Rapid EEG desynchronization and EMG activation induced by intravenous cocaine in freely moving rats: a peripheral, nondopamine neural triggering.

Many important physiological, behavioral, and psychoemotional effects of intravenous (IV) cocaine (COC) are too fast and transient compared with pharmacokinetic predictions, suggesting a possible involvement of peripheral neural mechanisms in their triggering. In the present study, we examined changes in cortical electroencephalogram (EEG) and neck electromyogram (EMG) induced in freely moving rats by IV COC administration at low, reinforcing doses (0.25-1.0 mg/kg) and compared them with those induced by an auditory stimulus and IV COC methiodide, which cannot cross the blood-brain barrier. We found that COC induces rapid, strong, and prolonged EEG desynchronization, associated with decrease in alpha and increase in beta and gamma activities, and EMG activation and that both begin within 2-6 s following the start of a 10-s injection; immediate components of this effect were dose independent. The rapid COC-induced changes in EEG and EMG resembled those induced by an auditory stimulus; the latter effects had shorter onset latencies and durations and were fully blocked during urethane anesthesia. Although urethane anesthesia completely blocked COC-induced EMG activation and rapid components of EEG response, COC still induced EEG desynchronization that was much weaker, greatly delayed (approximately 60 s), and associated with tonic decreases in delta and increases in alpha, beta, and gamma activities. Surprisingly, IV saline delivered during slow-wave sleep (but not quite wakefulness) also induced a transient EEG desynchronization but without changes in EMG activity; these effects were also fully blocked during anesthesia. Peripherally acting COC methiodide fully mimicked rapid EEG and EMG effects of regular COC, but the effects at an equimolar dose were less prolonged than those with regular COC. These data suggest that in awake animals IV COC, like somato-sensory stimuli, induces cortical activation and a subsequent motor response via its action on peripheral neural elements and involving rapid neural transmission. By providing a rapid neural signal and triggering transient neural activation, such an action might play a crucial role in the sensory effects of COC, thus contributing to the learning and development of drug-taking behavior.

Fluctuations in central and peripheral temperatures associated with feeding behavior in rats.

We examined the pattern of temperature fluctuations in the nucleus accumbens (NAcc), temporal muscle, and skin, along with locomotion in food-deprived and nondeprived rats following the presentation of an open or closed food container and during subsequent eating or food-seeking behavior without eating. Although rats in food-deprived, quiet resting conditions had more than twofold lower spontaneous locomotion and lower temperature values than in nondeprived conditions, after presentation of a container, they consistently displayed food-seeking behavior, showing much larger and longer temperature changes. When the container was open, rats rapidly retrieved food and consumed it. Food consumption was preceded and accompanied by gradual increases in brain and muscle temperatures ( approximately 1.5 degrees C) and a weaker, delayed increase in skin temperature ( approximately 0.8 degrees C). All temperatures began to rapidly fall immediately after eating was completed, but NAcc and muscle temperatures returned to baseline after approximately 35 min. When the container was closed and rats were unable to obtain food, they continued food-seeking activity during the entire period of presentation. Similar to eating, this activity was preceded and accompanied by gradual temperature increases in the brain and muscle, which were somewhat smaller than those during eating ( approximately 1.2 degrees C), with no changes in skin temperature. In contrast to trials with eating, NAcc and muscle temperatures continued to increase for approximately 10 min after the container was removed from the cage and the rat continued food-seeking behavior, with a return to baselines after approximately 50 min. These temperature fluctuations are discussed with respect to alterations in metabolic brain activity associated with feeding behavior, depending upon deprivation state and food availability.

Behavioral and temperature effects of delta 9-tetrahydrocannabinol in human-relevant doses in rats.

Marijuana smoking dramatically alters responses to various environmental stimuli. To study this phenomenon, we assessed how delta-9-tetrahydrocannabinol (THC), a primary psychoactive ingredient of marijuana, affects locomotor and brain (nucleus accumbens or NAcc), muscle and skin temperature responses to natural arousing stimuli (one-minute tail-pinch and one-minute social interaction with another male rat) and iv cocaine (1 mg/kg) in male rats. THC was administered at three widely varying doses (0.5, 2.0 and 8.0 mg/kg, ip), and the drug-induced changes in basal values and responses to stimuli were compared to those occurring following ip vehicle injections (control). Each stimulus in control conditions caused acute locomotor activation, a prolonged increase in brain and muscle temperature (0.6-1.0 degrees C for 20-50 min) and transient decrease in skin temperature (-0.6 degrees C for 1-3 min). While THC at any dose had a tendency to decrease spontaneous locomotion as well as brain and muscle temperatures, true hypothermia and hypoactivity as well as clearly diminished locomotor and temperature responses to all stimuli were only seen following the largest dose. In this case, temperature decreases in the NAcc were stronger than in the muscle, suggesting metabolic brain inhibition as the primary cause of hypoactivity, hypothermia and hyporesponsiveness. While weaker in strength and without associated vasodilatation, this response pattern is mimicked by general anesthetics, questioning to what extent the hypothermic action of THC is specific (i.e., mediated via endogenous cannabinoid receptors) or non-specific, reflecting drug interaction with membrane lipids or other receptors. In contrast, weaker behavioral and temperature effects of THC at lower doses resemble those of diazepam, whose locomotion- and temperature-decreasing effects are evident only in activated conditions, when rats are moving and basal temperatures are elevated.