SynapseJ: An Automated, Synapse Identification Macro for ImageJ

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.

Efficient Actor-Critic Reinforcement Learning With Embodiment of Muscle Tone for Posture Stabilization of the Human Arm

This letter proposes a new idea to improve learning efficiency in reinforcement learning (RL) with the actor-critic method used as a muscle controller for posture stabilization of the human arm. Actor-critic RL (ACRL) is used for simulations to realize posture controls in humans or robots using muscle tension control. However, it requires very high computational costs to acquire a better muscle control policy for desirable postures. For efficient ACRL, we focused on embodiment that is supposed to potentially achieve efficient controls in research fields of artificial intelligence or robotics. According to the neurophysiology of motion control obtained from experimental studies using animals or humans, the pedunculopontine tegmental nucleus (PPTn) induces muscle tone suppression, and the midbrain locomotor region (MLR) induces muscle tone promotion. PPTn and MLR modulate the activation levels of mutually antagonizing muscles such as flexors and extensors in a process through which control signals are translated from the substantia nigra reticulata to the brain stem. Therefore, we hypothesized that the PPTn and MLR could control muscle tone, that is, the maximum values of activation levels of mutually antagonizing muscles using different sigmoidal functions for each muscle; then we introduced antagonism function models (AFMs) of PPTn and MLR for individual muscles, incorporating the hypothesis into the process to determine the activation level of each muscle based on the output of the actor in ACRL. ACRL with AFMs representing the embodiment of muscle tone successfully achieved posture stabilization in five joint motions of the right arm of a human adult male under gravity in predetermined target angles at an earlier period of learning than the learning methods without AFMs. The results obtained from this study suggest that the introduction of embodiment of muscle tone can enhance learning efficiency in posture stabilization disorders of humans or humanoid robots.

Deep brain stimulation for Stroke: Continuous stimulation of the pedunculopontine tegmental nucleus has no impact on skilled walking in rats after photothrombotic stroke

Background: Gait impairment after stroke is considered as a loss of cerebral function but is also the result of dysfunctional cerebral signals travelling to the spinal motor centres. A therapeutic option to restore disturbed cerebral network activity is deep brain stimulation (DBS). Methods: A promising target for neuromodulation might be the pedunculopontine tegmental nucleus (PPTg), which contributes to the initiation and control of gait. To test this hypothesis, we trained eighteen rats to cross a horizontal ladder and a wooden beam before inflicting a photothrombosis in the right sensorimotor cortex and implanting a stimulating electrode in the ipsilateral PPTg. Results: Continuous high-frequency DBS (130 Hz; amplitude 55 ± 5 µA) of rats for 10 days yielded no significant improvement of skilled walking when examined with the ladder rung walking test and beam walking test compared to shamstimulation. Conclusion: In contrast to DBS of the cuneiform nucleus, PPTg-stimulation improves neither control of gait nor balance after stroke.

Efficacy of Deep Brain Stimulation on the Improvement of the Bladder Functions in Traumatic Brain Injured Rats

Objective: Traumatic brain injuries (TBIs) are a prime public health challenge with a high incidence of mortality, and also reflect severe economic impacts. One of their severe symptoms is bladder dysfunction. Conventional therapeutic methods are not effective in managing bladder dysfunction. Henceforth, a research endeavor was attempted to explore a new therapeutic approach for bladder dysfunction through deep brain stimulation (DBS) procedures in a TBI animal model. Methods: TBI in this animal model was induced by the weight-drop method. All rats with an induced TBI were housed for 4 weeks to allow severe bladder dysfunction to develop. Subsequently, an initial urodynamic measurement, the simultaneous recording of cystometric (CMG) and external urethral sphincter electromyography (EUS-EMG) activity was conducted to evaluate bladder function. Further, standard DBS procedures with varying electrical stimulation parameters were executed in the target area of the pedunculopontine tegmental nucleus (PPTg). Simultaneously, urodynamic measurements were re-established to compare the effects of DBS interventions on bladder functions. Results: From the variable combinations of electrical stimulation, DBS at 50 Hz and 2.0 V, significantly reverted the voiding efficiency from 39% to 69% in TBI rats. Furthermore, MRI studies revealed the precise localization of the DBS electrode in the target area. Conclusions: The results we obtained showed an insightful understanding of PPTg-DBS and its therapeutic applications in alleviating bladder dysfunction in rats with a TBI. Hence, the present study suggests that PPTg-DBS is an effective therapeutic strategy for treating bladder dysfunction.

Deep Brain Stimulation of the Pedunculopontine Tegmental Nucleus Renders Neuroprotection through the Suppression of Hippocampal Apoptosis: An Experimental Animal Study

The core objective of this study was to determine the neuroprotective properties of deep brain stimulation of the pedunculopontine tegmental nucleus on the apoptosis of the hippocampus. The pedunculopontine tegmental nucleus is a prime target for Parkinson′s disease and is a crucial component in a feedback loop connected with the hippocampus. Deep brain stimulation was employed as a potential tool to evaluate the neuroprotective properties of hippocampal apoptosis. Deep brain stimulation was applied to the experimental animals for an hour. Henceforth, the activity of Caspase-3, myelin basic protein, Bcl-2, BAX level, lipid peroxidation, interleukin-6 levels, and brain-derived neurotrophic factor levels were evaluated at hours 1, 3 and 6 and compared with the sham group of animals. Herein, decreased levels of caspases activity and elevated levels of Bcl-2 expressions and inhibited BAX expressions were observed in experimental animals at the aforementioned time intervals. Furthermore, the ratio of Bcl-2/BAX was increased, and interleukin -6, lipid peroxidation levels were not affected by deep brain stimulation in the experimental animals. These affirmative results have explained the neuroprotection rendered by hippocampus apoptosis as a result of deep brain stimulation. Deep brain stimulation is widely used to manage neuro-motor disorders. Nevertheless, this novel study will be a revelation for a better understanding of neuromodulatory management and encourage further research with new dimensions in the field of neuroscience.

Distinct pathways to the parafacial respiratory group to trigger active expiration in adult rats

Active expiration (AE) is part of the breathing phase; it is conditional and occurs when we increase our metabolic demand, such as during hypercapnia, hypoxia, or exercise. The parafacial respiratory group (pFRG) is involved in AE. Data from the literature suggest that excitatory and the absence of inhibitory inputs to the pFRG are necessary to determine AE. However, the source of the inputs to the pFRG that trigger AE remains unclear. We show in adult urethane-anesthetized Wistar rats that the pharmacological inhibition of the medial aspect of the nucleus of the solitary tract (mNTS) or the rostral aspect of the pedunculopontine tegmental nucleus (rPPTg) is able to generate AE. In addition, direct inhibitory projection from the mNTS or indirect cholinergic projection from the rPPTg is able to contact pFRG to trigger AE. The inhibition of the mNTS or the rPPTg under conditions of high metabolic demand, such as hypercapnia (9–10% CO2), did not affect the AE. The present results suggest for the first time that inhibitory sources from the mNTS and a cholinergic pathway from the rPPTg, involving M2/M4 muscarinic receptors, could be important sources to modulate and sustain AE.