Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2.

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5-mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2-ERK1/2 interactions.

The origin of the phrase comparative psychology: an historical overview.

Comparative psychology, in its narrow meaning, refers to the study of the similarities and differences in the psychology and behavior of different species. In a broader meaning, it includes comparisons between different biological and socio-cultural groups, such as species, sexes, developmental stages, ages, and ethnicities. This broader meaning originated by extension from the former narrow meaning, which historically was the original meaning of the phrase (interspecies psychological and behavioral comparisons) and which still today is the focus of the field. Currently, comparative psychology is a subject of study in hundreds of universities all over the world. Nevertheless, a question that is often asked but seldom answered is: when did the phrase comparative psychology first appear and where did it come from? In the present work, we tracked down the origins of the phrase comparative psychology. In order to do so, at first we described the origin of the word psychology, coined in the decade 1510-1520 in the Republic of Venice by Dalmatian Renaissance humanist Marko Marulic Splicanin (1450-1524). Then, to explain where, within the phrase comparative psychology, the term comparative came from, we outlined the origin of the use of the word comparative in reference to interspecies comparisons. Finally, we reported the origin of the combination of the words comparative and psychology to form the phrase comparative psychology, the first usage of which was in 1778 by German scholar Michael Hissmann (1752-1784) from the University of Göttingen. Origins of the phrase in Latin, German, French and English languages are described.

Visual Evoked Potentials to Monitor Myelin Cuprizone-Induced Functional Changes.

The visual system is one of the most accessible routes to study the central nervous system under pathological conditions, such as in multiple sclerosis (MS). Non-invasive visual evoked potential (VEP) and optical coherence tomography (OCT) were used to assess visual function and neuroretinal thickness in C57BL/6 taking 0.2% cuprizone for 7 weeks and at 5, 8, 12, and 15 days after returning to a normal diet. VEPs were significantly delayed starting from 4 weeks on cuprizone, with progressive recovery off cuprizone, becoming significant at day 8, complete at day 15. In contrast, OCT and neurofilament staining showed no significant axonal thinning. Optic nerve histology indicated that whilst there was significant myelin loss at 7 weeks on the cuprizone diet compared with healthy mice, at 15 days off cuprizone diet demyelination was significantly less severe. The number of Iba 1+ cells was found increased in cuprizone mice at 7 weeks on and 15 days off cuprizone. The combined use of VEPs and OCT allowed us to characterize non-invasively, in vivo, the functional and structural changes associated with demyelination and remyelination in a preclinical model of MS. This approach contributes to the non-invasive study of possible effective treatments to promote remyelination in demyelinating pathologies.

Dose-dependent effect of myelin oligodendrocyte glycoprotein on visual function and optic nerve damage in experimental autoimmune encephalomyelitis.

Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5 weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing-remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose-dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Non-invasive visual evoked potentials under sevoflurane versus ketamine-xylazine in rats.

BACKGROUND: Visual Evoked Potential (VEP) quantifies electrical signals produced in visual cortex in response to visual stimuli. VEP elicited by light flashes is a useful biomarker to evaluate visual function in preclinical models and it can be recorded in awake or anaesthetised state. Different types of anaesthesia influence VEP properties, such as latency, which measures the propagation speed along nerve fibers, and amplitude that quantifies the power of electrical signal. AIM: The goal of this work is to compare VEPs elicited in Dark Agouti rats under two types of anaesthesia: volatile sevoflurane or injectable ketamine-xylazine. METHODS: VEP latency, amplitude, signal-to-noise ratio and recording duration were measured in Dark Agouti rats randomly assigned to two groups, the first subjected to volatile sevoflurane and the second to injectable ketamine-xylazine. Taking advantage of non-invasive flash-VEP recording through epidermal cup electrodes, three time points of VEP recordings were assessed in two weeks intervals. RESULTS: VEP recorded under ketamine-xylazine showed longer latency and higher amplitude compared with sevoflurane, with analogous repeatability over time. However, sevoflurane tended to suppress electrical signals from visual cortex, resulting in a lower signal-to-noise ratio. Moreover, VEP procedure duration lasted longer in rats anaesthetised with sevoflurane than ketamine-xylazine. CONCLUSIONS: In Dark Agouti rats, the use of different anaesthesia can influence VEP components in terms of latency and amplitude. Notably, sevoflurane and ketamine-xylazine revealed satisfying repeatability over time, which is critical to perform reliable follow-up studies. Ketamine-xylazine allowed to obtain more clearly discernible VEP components and less background noise, together with a quicker recording procedure and a consequently improved animal safety and welfare.

Apparatus design and behavioural testing protocol for the evaluation of spatial working memory in mice through the spontaneous alternation T-maze.

Spatial working memory can be assessed in mice through the spontaneous alternation T-maze test. The T-maze is a T-shaped apparatus featuring a stem (start arm) and two lateral goal arms (left and right arms). The procedure is based on the natural tendency of rodents to prefer exploring a novel arm over a familiar one, which induces them to alternate the choice of the goal arm across repeated trials. During the task, in order to successfully alternate choices across trials, an animal has to remember which arm had been visited in the previous trial, which makes spontaneous alternation T-maze an optimal test for spatial working memory. As this test relies on a spontaneous behaviour and does not require rewards, punishments or pre-training, it represents a particularly useful tool for cognitive evaluation, both time-saving and animal-friendly. We describe here in detail the apparatus and the protocol, providing representative results on wild-type healthy mice.

The 4-Hole-Board Test for Assessment of Long-Term Spatial Memory in Mice.

The hole-board test has been used in rodents since the early 60s to measure exploratory behavior, locomotor activity and cognitive function. The test is based on rodents' natural curiosity and attraction for novelty. Basically, the hole-board consists of a small square arena with an extractable platform as floor, which has a set of equally spaced circular holes on its surface. In this article, we describe the protocol of a 4-hole-board test allowing the assessment of long-term spatial memory in mice without the employment of water or food restriction, painful stimuli (as electrical shocks) or any aversive condition (as forced swimming or exposure to intense light). Four holes are present on the floor of the square arena (one for each of its four quadrants). Mice released in the arena spontaneously approach the holes and explore them by briefly inserting the snout inside, a behavior defined as nose-poking (or head-dipping). If, after 24 hr, rodents are re-exposed to the hole-board, the novelty of the holes decreases. Animals with an intact long-term memory will show a reduction of the frequency of nose-poking into the holes. The total number of nose-pokes on day 1 is an index of exploration, while the percentage of decrease in nose-poking on day 2 represents an index of long-term spatial memory. Number of quadrant crossings is scored as a control measure for locomotor activity, which with the present protocol should remain stable across the days of testing. Indeed, the 4-hole-board test represents a stress-free and animal-friendly option to evaluate long-term spatial memory. In the present paper, we provide detailed description of the hole-board apparatus and step-by-step protocol for assessment of spatial memory in mice. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Validation of the 4-hole-board Basic Protocol 2: Evaluation of long-term spatial memory through the 4-hole-board test.

Functional evolution of visual involvement in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a common animal model of multiple sclerosis (MS). C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein exhibit chronic disease course, together with optic neuritis, consisting of demyelination/axonal loss of the optic nerve. OBJECTIVES: To characterize functional and structural visual damages in two different phases of EAE: pre- and post-motor onset. METHODS: Visual alterations were detected with Visual Evoked Potential (VEP), Electroretinogram (ERG) and Optical Coherence Tomography (OCT). Optic nerve histology was performed at 7 (pre-motor onset) or 37 (post-motor onset) days post-immunization (dpi). RESULTS: At 7 dpi, optic nerve inflammation was similar in EAE eyes with and without VEP latency delay. Demyelination was detected in EAE eyes with latency delay (p < 0.0001), while axonal loss (p < 0.0001) and ERG b-wave amplitude (p = 0.004) were decreased in EAE eyes without latency delay compared to Healthy controls. At 37 dpi, functional and structural optic nerve damage were comparable between EAE groups, while a decrease of ERG amplitude and NGCC thickness were found in EAE eyes with VEP latency delay detected post-motor onset. CONCLUSIONS: Thanks to non-invasive methods, we studied the visual system in a MS model, which could be useful for developing specific therapeutic strategies to target different disease phases.

Non-invasive visual evoked potentials to assess optic nerve involvement in the dark agouti rat model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein.

Experimental autoimmune encephalomyelitis (EAE) is the primary disease model of multiple sclerosis (MS), one of the most diffused neurological diseases characterized by fatigue, muscle weakness, vision loss, anxiety and depression. EAE can be induced through injection of myelin peptides to susceptible mouse or rat strains. In particular, EAE elicited by the autoimmune reaction against myelin oligodendrocyte glycoprotein (MOG) presents the common features of human MS: inflammation, demyelination and axonal loss. Optic neuritis affects visual pathways in both MS and in several EAE models. Neurophysiological evaluation through visual evoked potential (VEP) recording is useful to check visual pathway dysfunctions and to test the efficacy of innovative treatments against optic neuritis. For this purpose, we investigate the extent of VEP abnormalities in the dark agouti (DA) rat immunized with MOG, which develops a relapsing-remitting disease course. Together with the detection of motor signs, we acquired VEPs during both early and late stages of EAE, taking advantage of a non-invasive recording procedure that allows long follow-up studies. The validation of VEP outcomes was determined by comparison with ON histopathology, aimed at revealing inflammation, demyelination and nerve fiber loss. Our results indicate that the first VEP latency delay in MOG-EAE DA rats appeared before motor deficits and were mainly related to an inflammatory state. Subsequent VEP delays, detected during relapsing EAE phases, were associated with a combination of inflammation, demyelination and axonal loss. Moreover, DA rats with atypical EAE clinical course tested at extremely late time points, manifested abnormal VEPs although motor signs were mild. Overall, our data demonstrated that non-invasive VEPs are a powerful tool to detect visual involvement at different stages of EAE, prompting their validation as biomarkers to test novel treatments against MS optic neuritis.

A new electrophysiological non-invasive method to assess retinocortical conduction time in the Dark Agouti rat through the simultaneous recording of electroretinogram and visual evoked potential.

PURPOSE: To develop a non-invasive method exploiting simultaneous recording of epidermal visual evoked potential (VEP) and epicorneal electroretinogram (ERG) to study retinocortical function and to evaluate its reliability and repeatability over time. METHODS: Female wild-type DA rats were anesthetized with ketamine/xylazine (40/5 mg/kg). Epidermal VEP (Ag/AgCl cup electrode on scalp) and epicorneal ERG (gold ring electrode on eye surface) were recorded simultaneously in response to flash stimulation. RESULTS: ANOVA for repeated measures showed that peak times of ERG b-wave and of VEP N1 and P2 were stable across 6 weekly time-points, as well as the corresponding amplitudes. Mean retinocortical time from b-wave to N1 (RCT1) was 7.6 ms and remained comparable across the 6 time-points. Mean retinocortical time from b-wave to P2 (RCT2) was 28.7 ms and did not show significant variations over time. Coefficient of variation (CoV%) and CoV% adjusted for sample size, namely relative standard error (RSE%), were calculated as indexes of repeatability. Good RSE% over time was obtained (< 5% for b-wave, N1 and P2 peak times; < 20% and < 7% for RCT1 and RCT2, respectively). CONCLUSIONS: Simultaneous recording of ERG and VEP has been previously achieved through invasive methods requiring surgery. Here, we present a new non-invasive method, which allowed to obtain peak and retinocortical times that were constant across a long period and had a good repeatability over time. This method will ensure not only a gain in animal welfare, but will also avoid stress and eye or brain lesions which can interfere with experimental variables.

Semi-invasive and non-invasive recording of visual evoked potentials in mice.

PURPOSE: Visual evoked potentials (VEPs) are used to assess visual function in preclinical models of neurodegenerative diseases. VEP recording with epidural screw electrodes is a common method to study visual function in rodents, despite being an invasive procedure that can damage the tissue under the skull. The present study was performed to test a semi-invasive (epicranial) and a non-invasive (epidermal) VEP recording technique, comparing them with the classic epidural acquisition method. METHODS: Flash VEPs were recorded from C57BL/6 mice on three separate days within 2 weeks. Waveforms, latencies and amplitudes of the components were compared between the three different methods, utilizing coefficient of repeatability, coefficient of variation and intersession standard deviation to evaluate reproducibility. RESULTS: While epidural electrodes succeeded in recording two negative peaks (N1 and N2), epicranial and epidermal electrodes recorded a single peak (N1). Statistical indexes showed a comparable reproducibility between the three techniques, with a greater stability of N1 latency recorded through epicranial electrodes. Moreover, N1 amplitudes recorded with the new less-invasive methods were more reproducible compared to the invasive gold-standard technique. CONCLUSIONS: These results demonstrate the reliability of semi- and non-invasive VEP recordings, which can be useful to evaluate murine models of neurological diseases.

Optic nerve involvement in experimental autoimmune encephalomyelitis to homologous spinal cord homogenate immunization in the dark agouti rat.

Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis. We assessed motor signs and recorded VEPs for five or eight weeks with epidural or epidermal electrodes, respectively, with final histopathology of optic nerves (ONs). Injected rats exhibited motor deficits a week after immunization. VEP delays arose from the 2nd to the 5th week, when a recovery occurred in epidermal-recorded rats. ON damage appeared in epidural-, but not in epidermal-recorded rats, probably due to a remyelination process. VEP could be exploited as neurophysiological marker to test novel treatments against neurodegeneration involving ONs.

Visual evoked potentials can be reliably recorded using noninvasive epidermal electrodes in the anesthetized rat.

PURPOSE: Visual evoked potentials (VEPs) are a powerful tool to evaluate nervous conduction along the visual pathways, both in humans and in animal models. Traditionally, epidural screw electrodes are used to record VEPs in preclinical research. Here we tested the feasibility in the preclinical setting of the same noninvasive technique used for clinical VEP acquisition, by using epidermal cup electrodes with no surgical procedures. METHODS: Monocular flash VEPs were recorded bilaterally under sevoflurane anesthesia once a week for 6 weeks in 14 dark Agouti rats, 7 with implanted epidural screws and 7 with epidermal 6 mm Ø Ag/AgCl cups. RESULTS: VEP traces obtained with the two techniques were morphologically comparable. There were no significant differences in latency of the main visual component between screw-recorded VEPs (sVEPs) and cup-recorded VEPs (cVEPs). Amplitude values with epidermal cups were significantly lower than those with epidural screws. Both techniques provided latencies and amplitudes which were stable over time. Furthermore, with regard to latency both methods ensured highly repeatable measurements over time, with epidermal cups even providing slightly better results. On the other hand, considering amplitudes, cVEPs and sVEPs provided fairly acceptable repeatability. CONCLUSIONS: Epidermal cup electrodes can provide comparable results to those obtained with the "gold standard" epidural screws, while representing a simpler and less invasive technique to test nervous conduction along the visual pathways in the preclinical setting.

Severe Intellectual Disability and Enhanced Gamma-Aminobutyric Acidergic Synaptogenesis in a Novel Model of Rare RASopathies.

BACKGROUND: Dysregulation of Ras-extracellular signal-related kinase (ERK) signaling gives rise to RASopathies, a class of neurodevelopmental syndromes associated with intellectual disability. Recently, much attention has been directed at models bearing mild forms of RASopathies whose behavioral impairments can be attenuated by inhibiting the Ras-ERK cascade in the adult. Little is known about the brain mechanisms in severe forms of these disorders. METHODS: We performed an extensive characterization of a new brain-specific model of severe forms of RASopathies, the KRAS12V mutant mouse. RESULTS: The KRAS12V mutation results in a severe form of intellectual disability, which parallels mental deficits found in patients bearing mutations in this gene. KRAS12V mice show a severe impairment of both short- and long-term memory in a number of behavioral tasks. At the cellular level, an upregulation of ERK signaling during early phases of postnatal development, but not in the adult state, results in a selective enhancement of synaptogenesis in gamma-aminobutyric acidergic interneurons. The enhancement of ERK activity in interneurons at this critical postnatal time leads to a permanent increase in the inhibitory tone throughout the brain, manifesting in reduced synaptic transmission and long-term plasticity in the hippocampus. In the adult, the behavioral and electrophysiological phenotypes in KRAS12V mice can be temporarily reverted by inhibiting gamma-aminobutyric acid signaling but not by a Ras-ERK blockade. Importantly, the synaptogenesis phenotype can be rescued by a treatment at the developmental stage with Ras-ERK inhibitors. CONCLUSIONS: These data demonstrate a novel mechanism underlying inhibitory synaptogenesis and provide new insights in understanding mental dysfunctions associated to RASopathies.

Behavioral methods for the study of the Ras-ERK pathway in memory formation and consolidation: passive avoidance and novel object recognition tests.

Memory is a high-level brain function that enables organisms to adapt their behavioral responses to the environment, hence increasing their probability of survival. The Ras-ERK pathway is a key molecular intracellular signalling cascade for memory consolidation. In this chapter we will describe two main one-trial behavioral tests commonly used in the field of memory research in order to assess the role of Ras-ERK signalling in long-term memory: passive avoidance and object recognition. Passive avoidance (PA) is a fear-motivated instrumental learning task, designed by Jarvik and Essman in 1960, in which animals learn to refrain from emitting a behavioral response that has previously been associated with a punishment. We will describe here the detailed protocol and show some examples of how PA can reveal impairments or enhancements in memory consolidation following loss or gain of function genetic manipulations of the Ras-ERK pathway. The phenotypes of global mutants as Ras-GRF1 KO, GENA53, and ERK1 KO mice, as well as of conditional region-specific mutants (striatal K-CREB mice), will be illustrated as examples. Novel object recognition (NOR), developed by Ennaceur and Delacour in 1988, is instead a more recent and highly ecological test, which relies on the natural tendency of rodents to spontaneously approach and explore novel objects, representing hence a useful non-stressful tool for the study of memory in animals without the employment of punishments or starvation/water restriction regimens. Careful indications will be given on how to select the positions for the novel object, in order to counterbalance for individual side preferences among mice during the training. Finally, the methods for calculating two learning indexes will be described. In addition to the classical discrimination index (DI) that measures the ability of an animal to discriminate between two different objects which are presented at the same time, we will describe the formula of a new index that we present here for the first time, the recognition index (RI), which quantifies the ability of an animal to recognize a same object at different time points and that, by taking into account the basal individual preferences displayed during the training, can give a more accurate measure of an animal's actual recognition memory.

Molecular and cellular mechanisms of dopamine-mediated behavioral plasticity in the striatum.

The striatum is the input structure of the basal ganglia system. By integrating glutamatergic signals from cortical and subcortical regions and dopaminergic signals from mesolimbic nuclei the striatum functions as an important neural substrate for procedural and motor learning as well as for reward-guided behaviors. In addition, striatal activity is significantly altered in pathological conditions in which either a loss of dopamine innervation (Parkinson's disease) or aberrant dopamine-mediated signaling (drug addiction and L-DOPA induced dyskinesia) occurs. Here we discuss cellular mechanisms of striatal synaptic plasticity and aspects of cell signaling underlying striatum-dependent behavior, with a major focus on the neuromodulatory action of the endocannabinoid system and on the role of the Ras-ERK cascade.

Mice Lacking Ras-GRF1 Show Contextual Fear Conditioning but not Spatial Memory Impairments: Convergent Evidence from Two Independently Generated Mouse Mutant Lines.

Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning Brambilla's mice with a third mouse line (GENA53) in which a non-sense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in Brambilla's mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdala functions but possibly to some distinct hippocampal connections specific to contextual learning.

Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model.

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing.