Visual training after central retinal loss limits structural white matter degradation: an MRI study.

BACKGROUND: Macular degeneration of the eye is a common cause of blindness and affects 8% of the worldwide human population. In adult cats with bilateral lesions of the central retina, we explored the possibility that motion perception training can limit the associated degradation of the visual system. We evaluated how visual training affects behavioral performance and white matter structure. Recently, we proposed (Kozak et al. in Transl Vis Sci Technol 10:9, 2021) a new motion-acuity test for low vision patients, enabling full visual field functional assessment through simultaneous perception of shape and motion. Here, we integrated this test as the last step of a 10-week motion-perception training. RESULTS: Cats were divided into three groups: retinal-lesioned only and two trained groups, retinal-lesioned trained and control trained. The behavioral data revealed that trained cats with retinal lesions were superior in motion tasks, even when the difficulty relied only on acuity. 7 T-MRI scanning was done before and after lesioning at 5 different timepoints, followed by Fixel-Based and Fractional Anisotropy Analysis. In cats with retinal lesions, training resulted in a more localized and reduced percentage decrease in Fixel-Based Analysis metrics in the dLGN, caudate nucleus and hippocampus compared to untrained cats. In motion-sensitive area V5/PMLS, the significant decreases in fiber density were equally strong in retinal-lesioned untrained and trained cats, up to 40% in both groups. The only cortical area with Fractional Anisotropy values not affected by central retinal loss was area V5/PMLS. In other visual ROIs, the Fractional Anisotropy values increased over time in the untrained retinal lesioned group, whereas they decreased in the retinal lesioned trained group and remained at a similar level as in trained controls. CONCLUSIONS: Overall, our MRI results showed a stabilizing effect of motion training applied soon after central retinal loss induction on white matter structure. We propose that introducing early motion-acuity training for low vision patients, aimed at the intact and active retinal peripheries, may facilitate brain plasticity processes toward better vision.

Preparation of High-Viability Single-Cell Suspensions from African Turquoise Killifish Brain Tissue.

Studying the brain at the single-cell level has become increasingly popular in recent years. This, however, remains challenging, especially in emerging model organisms. To carry out single-cell sequencing, the preparation of a high-viability single-cell suspension is critical. In this protocol, we describe how to prepare a high-viability single-cell suspension starting from brain tissue of the African turquoise killifish (Nothobranchius furzeri). The protocol consists of dissection, enzymatic and mechanical dissociation of the brain tissue, and debris removal. The protocol described here has been successfully used for 10× Genomics single-cell sequencing of the telencephalon of adult killifish, which requires a cell viability of at least 70%. In addition to single-cell sequencing experiments, the single-cell suspension generated can be used for other applications, including cell culture and flow cytometry.

A Robust and Reproducible Method to Study Neurorepair after Stab Injury in the African Turquoise Killifish Telencephalon.

The aging population (people >60 yr old) is steadily increasing worldwide, resulting in an increased prevalence of age-related neurodegenerative diseases. Despite intensive research efforts in the past decades, there are still no therapies available to stop, cure, or prevent these diseases. Induction of successful neuroregeneration (i.e., the production of new neurons that can functionally integrate into the existing neural circuitry) could represent a therapy to replace neurons lost by injury or disease in the aged central nervous system. The African turquoise killifish, with its particularly short life span, has emerged as a useful model to study how aging influences neuroregeneration. Here, we describe a robust and reproducible stab-injury protocol to study regeneration in the telencephalon of the African turquoise killifish. After the injury, newborn cells are traced by conducting a BrdU pulse-chase experiment. To identify newborn neurons, a double immunohistochemical staining for BrdU and HuCD is carried out. Techniques such as bromodeoxyuridine (BrdU) labeling, intracardial perfusion, cryosectioning, and immunofluorescence staining are described as separate sections.

Chemogenetic manipulation of astrocyte activity at the synapse- a gateway to manage brain disease.

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.

A short dasatinib and quercetin treatment is sufficient to reinstate potent adult neuroregenesis in the aged killifish.

The young African turquoise killifish has a high regenerative capacity, but loses it with advancing age, adopting several aspects of the limited form of mammalian regeneration. We deployed a proteomic strategy to identify pathways that underpin the loss of regenerative power caused by aging. Cellular senescence stood out as a potential brake on successful neurorepair. We applied the senolytic cocktail Dasatinib and Quercetin (D + Q) to test clearance of chronic senescent cells from the aged killifish central nervous system (CNS) as well as rebooting the neurogenic output. Our results show that the entire aged killifish telencephalon holds a very high senescent cell burden, including the parenchyma and the neurogenic niches, which could be diminished by a short-term, late-onset D + Q treatment. Reactive proliferation of non-glial progenitors increased substantially and lead to restorative neurogenesis after traumatic brain injury. Our results provide a cellular mechanism for age-related regeneration resilience and a proof-of-concept of a potential therapy to revive the neurogenic potential in an already aged or diseased CNS.

Analysis of Immediate Early Gene Expression Levels to Interrogate Changes in Cortical Neuronal Activity Patterns upon Vision Loss.

Mapping immediate early gene (IEG) expression levels to characterize changes in neuronal activity patterns has become a golden standard in neuroscience research. Due to straightforward detection methods such as in situ hybridization and immunohistochemistry, changes in IEG expression can be easily visualized across brain regions and in response to physiological and pathological stimulation. Based on in-house experience and existing literature, zif268 represents itself as the IEG of choice to investigate the neuronal activity dynamics induced by sensory deprivation. In the monocular enucleation mouse model of partial vision loss, zif268 in situ hybridization can be implemented to study cross-modal plasticity by charting the initial decline and subsequent rise in neuronal activity in visual cortical territory deprived of direct retinal visual input. Here, we describe a protocol for high-throughput radioactive zif268 in situ hybridization as a readout for cortical neuronal activity dynamics in response to partial vision loss in mice.

Age-related alterations in the behavioral response to a novel environment in the African turquoise killifish (Nothobranchius furzeri).

The African turquoise killifish (Nothobranchius furzeri) has emerged as a popular model organism for neuroscience research in the last decade. One of the reasons for its popularity is its short lifespan for a vertebrate organism. However, little research has been carried out using killifish in behavioral tests, especially looking at changes in their behavior upon aging. Therefore, we used the open field and the novel tank diving test to unravel killifish locomotion, exploration-related behavior, and behavioral changes over their adult lifespan. The characterization of this behavioral baseline is important for future experiments involving pharmacology to improve the aging phenotype. In this study, two cohorts of fish were used, one cohort was tested in the open field test and one cohort was tested in the novel tank diving test. Each cohort was tested from the age of 6 weeks to the age of 24 weeks and measurements were performed every three weeks. In the open field test, we found an increase in the time spent in the center zone from 18 weeks onward, which could indicate altered exploration behavior. However, upon aging, the fish also showed an increased immobility frequency and duration. In addition, after the age of 15 weeks, their locomotion decreased. In the novel tank diving test, we did not observe this aging effect on locomotion or exploration. Killifish spent around 80% of their time in the bottom half of the tank, and we could not observe habituation effects, indicating slow habituation to novel environments. Moreover, we observed that killifish showed homebase behavior in both tests. These homebases are mostly located near the edges of the open field test and at the bottom of the novel tank diving test. Altogether, in the open field test, the largest impact of aging on locomotion and exploration was observed beyond the age of 15 weeks. In the novel tank diving test, no effect of age was found. Therefore, to test the effects of pharmacology on innate behavior, the novel tank diving test is ideally suited because there is no confounding effect of aging.

Lifespan extension with preservation of hippocampal function in aged system xc--deficient male mice.

The cystine/glutamate antiporter system xc- has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT-/-) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT-/- mice led to the hypothesis that system xc- deletion would negatively affect life- and healthspan. Still, till now the role of system xc- in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT-/- mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT-/- mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT-/- mice. Targeting system xc- is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Endocannabinoids and cortical plasticity: CB1R as a possible regulator of the excitation/inhibition balance in health and disease.

The endocannabinoid system has been linked to neurological disorders in which the excitation inhibition (E/I) balance in the neocortex is dysregulated, such as schizophrenia. The main endocannabinoid receptor type 1 of the central nervous system-CB1R-is expressed on different cell types, that when activated, modulate the cortical E/I balance. Here we review how CB1R signalling contributes to phases of heightened plasticity of the neocortex. We review the major role of the CB1R in cortical plasticity throughout life, including the early life sensory critical periods, the later maturation phase of the association cortex in adolescence, and the adult phase of sensory deprivation-induced cortical plasticity. Endocannabinoid-mediated long-term potentiation and depression of synapse strength fine-tune the E/I balance in visual, somatosensory and association areas. We emphasize how a distinct set of key endocannabinoid-regulated elements such as GABA and glutamate release, basket parvalbumin interneurons, somatostatin interneurons and astrocytes, are essential for normal cortical plasticity and dysregulated in schizophrenia. Even though a lot of data has been gathered, mechanistic knowledge about the exact CB1R-based modulation of excitation and/or inhibition is still lacking depending on cortical area and maturation phase in life. We emphasize the importance of creating such detailed knowledge for a better comprehension of what underlies the dysregulation of the neocortex in schizophrenic patients in adulthood. We propose that taking age, brain area and cell type into consideration when modulating the cortical E/I imbalance via cannabinoid-based pharmacology may pave the way for better patient care.

Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri.

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration-competent vertebrate that evolved to naturally age extremely fast. Stab-wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non-glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.

A Fair Assessment of Evaluation Tools for the Murine Microbead Occlusion Model of Glaucoma.

Despite being one of the most studied eye diseases, clinical translation of glaucoma research is hampered, at least in part, by the lack of validated preclinical models and readouts. The most popular experimental glaucoma model is the murine microbead occlusion model, yet the observed mild phenotype, mixed success rate, and weak reproducibility urge for an expansion of available readout tools. For this purpose, we evaluated various measures that reflect early onset glaucomatous changes in the murine microbead occlusion model. Anterior chamber depth measurements and scotopic threshold response recordings were identified as an outstanding set of tools to assess the model's success rate and to chart glaucomatous damage (or neuroprotection in future studies), respectively. Both are easy-to-measure, in vivo tools with a fast acquisition time and high translatability to the clinic and can be used, whenever judged beneficial, in combination with the more conventional measures in present-day glaucoma research (i.e., intraocular pressure measurements and post-mortem histological analyses). Furthermore, we highlighted the use of dendritic arbor analysis as an alternative histological readout for retinal ganglion cell density counts.

Modeling Neuroregeneration and Neurorepair in an Aging Context: The Power of a Teleost Model.

Aging increases the risk for neurodegenerative disease and brain trauma, both leading to irreversible and multifaceted deficits that impose a clear societal and economic burden onto the growing world population. Despite tremendous research efforts, there are still no treatments available that can fully restore brain function, which would imply neuroregeneration. In the adult mammalian brain, neuroregeneration is naturally limited, even more so in an aging context. In view of the significant influence of aging on (late-onset) neurological disease, it is a critical factor in future research. This review discusses the use of a non-standard gerontology model, the teleost brain, for studying the impact of aging on neurorepair. Teleost fish share a vertebrate physiology with mammals, including mammalian-like aging, but in contrast to mammals have a high capacity for regeneration. Moreover, access to large mutagenesis screens empowers these teleost species to fill the gap between established invertebrate and rodent models. As such, we here highlight opportunities to decode the factor age in relation to neurorepair, and we propose the use of teleost fish, and in particular killifish, to fuel new research in the neuro-gerontology field.

Star power: the emerging role of astrocytes as neuronal partners during cortical plasticity.

Plasticity is a fundamental property of neuronal circuits, allowing them to adapt to alterations in activation. Generally speaking, plasticity has been viewed from a 'neuron-centric' perspective, with changes in circuit function attributed to alterations in neuronal excitability, synaptic strength or neuronal connectivity. However, it is now clear that glial cells, in particular astrocytes, are key regulators of neuronal plasticity. This article reviews recent progress made in understanding astrocyte function and attempts to summarize these functions into a coherent framework that positions astrocytes as central players in the plasticity process.

Posterior parietal cortex contributions to cross-modal brain plasticity upon sensory loss.

Sensory loss causes compensatory behavior, like echolocation upon vision loss or improved visual motion detection upon deafness. This is enabled by recruitment of the deprived cortical area by the intact senses. Such cross-modal plasticity can however hamper rehabilitation via sensory substitution devices. To steer rehabilitation towards the desired outcome for the patient, having control over the cross-modal take-over is essential. Evidence accumulates to support a role for the posterior parietal cortex (PPC) in multimodal plasticity. This area shows increased activity after sensory loss, keeping similar functions but driven by other senses. Patient-specific factors like stress, social situation, age and attention, have a significant influence on the PPC and on cross-modal plasticity. We propose that understanding the response of the PPC to sensory loss and context is extremely important for determining the best possible implant-based therapies, and that mouse research holds potential to help unraveling the underlying anatomical, cellular and neuromodulatory mechanisms.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Reconsidering the Border between the Visual and Posterior Parietal Cortex of Mice.

The posterior parietal cortex (PPC) contributes to multisensory and sensory-motor integration, as well as spatial navigation. Based on primate studies, the PPC is composed of several subdivisions with differing connection patterns, including areas that exhibit retinotopy. In mice the composition of the PPC is still under debate. We propose a revised anatomical delineation in which we classify the higher order visual areas rostrolateral area (RL), anteromedial area (AM), and Medio-Medial-Anterior cortex (MMA) as subregions of the mouse PPC. Retrograde and anterograde tracing revealed connectivity, characteristic for primate PPC, with sensory, retrosplenial, orbitofrontal, cingulate and motor cortex, as well as with several thalamic nuclei and the superior colliculus in the mouse. Regarding cortical input, RL receives major input from the somatosensory barrel field, while AM receives more input from the trunk, whereas MMA receives strong inputs from retrosplenial, cingulate, and orbitofrontal cortices. These input differences suggest that each posterior PPC subregion may have a distinct function. Summarized, we put forward a refined cortical map, including a mouse PPC that contains at least 6 subregions, RL, AM, MMA and PtP, MPta, LPta/A. These anatomical results set the stage for a more detailed understanding about the role that the PPC and its subdivisions play in multisensory integration-based behavior in mice.

c-Fos expression following context conditioning and deep brain stimulation in the bed nucleus of the stria terminalis in rats.

Deep brain stimulation (DBS) in the bed nucleus of the stria terminalis (BST), a region implicated in the expression of anxiety, shows promise in psychiatric patients, but its effects throughout the limbic system are largely unknown. In male Wistar rats, we first evaluated the neural signature of contextual fear (N = 16) and next, of the anxiolytic effects of high-frequency electrical stimulation in the BST (N = 31), by means of c-Fos protein expression. In non-operated animals, we found that the left medial anterior BST displayed increased c-Fos expression in anxious (i.e., context-conditioned) versus control subjects. Moreover, control rats showed asymmetric expression in the basolateral amygdala (BLA) (i.e., higher intensities in the right hemisphere), which was absent in anxious animals. The predominant finding in rats receiving bilateral BST stimulation was a striking increase in c-Fos expression throughout much of the left hemisphere, which was not confined to the predefined regions of interest. To conclude, we found evidence for lateralized c-Fos expression during the expression of contextual fear and anxiolytic high-frequency electrical stimulation of the BST, particularly in the medial anterior BST and BLA. In addition, we observed an extensive and unexpected left-sided c-Fos spread following bilateral stimulation in the BST.

Astrocytes shape the plastic response of adult cortical neurons to vision loss.

Astrocytes are vital for preserving correct brain functioning by continuously sustaining neuronal activity and survival. They are in contact with multiple synapses at once allowing the expansion of local synaptic events into activity changes in neuronal networks. Furthermore, cortical astrocytes integrate local sensory inputs and behavioral state. From an anatomical, molecular, and functional perspective, astrocytes are thus ideal candidates to influence complex large-scale brain mechanisms such as plasticity. We collected evidence for the astrocytic potential for plasticity modulation, using the monocular enucleation (ME) mouse model of visual cortex plasticity. The impact of one-eyed vision involves the functional recruitment of the deprived visual cortex by the spared senses within a 7-week time frame, reflecting a substantial change in sensory information processing. In visually deprived cortex, a swift upregulation in Aldh1l1-positive astrocyte density lasts until maximal functional recovery is reached. Transient metabolic silencing of visual cortex astrocytes at the time of ME induction, through intracranial fluorocitrate injections, reveals that astrocytes are required on site to achieve adequate long-term neuronal reactivation. In addition, chronic stimulation by Gi but not Gq G-protein coupled receptor activation of local astrocytes boosts the cortical plasticity phenomenon. Hence, functional manipulation of protoplasmic astrocytes has long-lasting effects on the functional recovery of cortical neurons upon sensory loss, possibly by influencing the neuronal threshold to reactivate. Together, our results highlight an integral role for astrocytes in mediating adult cortical plasticity and unmask astrocyte specific Gi signaling as an interesting therapeutic pathway for brain plasticity regulation.

It takes two: Bilateral bed nuclei of the stria terminalis mediate the expression of contextual fear, but not of moderate cued fear.

A growing body of research supports a prominent role for the bed nucleus of the stria terminalis (BST) in the expression of adaptive and perhaps even pathological anxiety. The traditional premise that the BST is required for long-duration responses to threats, but not for fear responses to distinct, short-lived cues may, however, be oversimplified. A thorough evaluation of the involvement of the BST in cued and contextual fear is therefore warranted. In a series of preregistered experiments using male Wistar rats, we first addressed the involvement of the BST in cued fear. Following up on earlier work where we found that BST lesions disrupted auditory fear while the animals were in a rather high stress state, we here show that the BST is not required for the expression of more specific fear for the tone under less stressful conditions. In the second part, we corroborate that the same lesion method does attenuate contextual fear. Furthermore, despite prior indications for an asymmetric recruitment of the BST during the expression of anxiety, we found that bilateral lesioning of the BST is required for a significant attenuation of the expression of contextual fear. A functional BST in only one hemisphere resulted in increased variability in the behavioral outcome. We conclude that, in animals that acquired a fear memory with an intact brain, the bilateral BST mediates the expression of contextual fear, but not of unambiguous cued fear.

Optogenetic Stimulation of the Superior Colliculus Confers Retinal Neuroprotection in a Mouse Glaucoma Model.

Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) in the eye, which ultimately results in visual impairment or even blindness. Because current therapies often fail to halt disease progression, there is an unmet need for novel neuroprotective therapies to support RGC survival. Various research lines suggest that visual target centers in the brain support RGC functioning and survival. Here, we explored whether increasing neuronal activity in one of these projection areas could improve survival of RGCs in a mouse glaucoma model. Prolonged activation of an important murine RGC target area, the superior colliculus (SC), was established via a novel optogenetic stimulation paradigm. By leveraging the unique channel kinetics of the stabilized step function opsin (SSFO), protracted stimulation of the SC was achieved with only a brief light pulse. SSFO-mediated collicular stimulation was confirmed by immunohistochemistry for the immediate-early gene c-Fos and behavioral tracking, which both demonstrated consistent neuronal activity upon repeated stimulation. Finally, the neuroprotective potential of optogenetic collicular stimulation was investigated in mice of either sex subjected to a glaucoma model and a 63% reduction in RGC loss was found. This work describes a new paradigm for optogenetic collicular stimulation and a first demonstration that increasing target neuron activity can increase survival of the projecting neurons.SIGNIFICANCE STATEMENT Despite glaucoma being a leading cause of blindness and visual impairment worldwide, no curative therapies exist. This study describes a novel paradigm to reduce retinal ganglion cell (RGC) degeneration underlying glaucoma. Building on previous observations that RGC survival is supported by the target neurons to which they project and using an innovative optogenetic approach, we increased neuronal activity in the mouse superior colliculus, a main projection target of rodent RGCs. This proved to be efficient in reducing RGC loss in a glaucoma model. Our findings establish a new optogenetic paradigm for target stimulation and encourage further exploration of the molecular signaling pathways mediating retrograde neuroprotective communication.