Hyperactive MEK1 Signaling in Cortical GABAergic Neurons Promotes Embryonic Parvalbumin Neuron Loss and Defects in Behavioral Inhibition.

Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes.

The Noonan Syndrome-linked Raf1L613V mutation drives increased glial number in the mouse cortex and enhanced learning.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.

Characterizing the effects of tonic 17ß-estradiol administration on spatial learning and memory in the follicle-deplete middle-aged female rat.

17ß-estradiol (E2)-containing hormone therapy is a safe, effective way to alleviate unwanted menopause symptoms. Preclinical research has focused upon the role of E2 in learning and memory using a surgically menopausal rodent model whereby the ovaries are removed. Given that most women retain their reproductive tract and undergo a natural menopause transition, it is necessary to understand how exogenous E2 impacts a structurally intact, but follicle-deplete, system. In the current study, 8 month old female rats were administered the ovatoxin 4-vinylcyclohexene diepoxide (VCD), which accelerates ovarian follicular depletion, to model the human menopause transition. After follicular depletion, at 11 months old, rats were administered Vehicle or tonic E2 treatment for 12 days prior to behavioral evaluation on spatial working and reference memory tasks. Results demonstrated that E2 had both enhancing and impairing effects on taxed working memory depending upon the learning or retention phases of the water radial-arm maze, with no impact on reference memory. Relationships between memory scores and circulating estrogen levels were specific to follicle-depleted rats without E2 treatment. Collectively, findings demonstrate the complexity of E2 administration in a follicle-depleted background, with cognitive effects specific to working memory; furthermore, E2 administration altered circulating hormonal milieu and relationships between hormone profiles and memory. In sum, menopausal etiology impacts the parameters of E2 effects on cognition, complementing prior work with other estrogen compounds. Deciphering estrogenic actions in a system wherein the reproductive tract remains intact with follicle-depleted ovaries, thus modeling the majority or menopausal women, is critical for translational perspectives.

Cognitive changes across the menopause transition: A longitudinal evaluation of the impact of age and ovarian status on spatial memory.

Cognitive changes that occur during mid-life and beyond are linked to both aging and the menopause transition. Studies in women suggest that the age at menopause onset can impact cognitive status later in life; yet, little is known about memory changes that occur during the transitional period to the postmenopausal state. The 4-vinylcyclohexene diepoxide (VCD) model simulates transitional menopause in rodents by depleting the immature ovarian follicle reserve and allowing animals to retain their follicle-deplete ovarian tissue, resulting in a profile similar to the majority of perimenopausal women. Here, Vehicle or VCD treatment was administered to ovary-intact adult and middle-aged Fischer-344 rats to assess the trajectory of cognitive change across time with normal aging and aging with transitional menopause via VCD-induced follicular depletion, as well as to evaluate whether age at the onset of follicular depletion plays a role in cognitive outcomes. Animals experiencing the onset of menopause at a younger age exhibited impaired spatial memory early in the transition to a follicle-deplete state. Additionally, at the mid- and post- follicular depletion time points, VCD-induced follicular depletion amplified an age effect on memory. Overall, these findings suggest that age at the onset of menopause is a critical parameter to consider when evaluating learning and memory across the transition to reproductive senescence. From a translational perspective, this study illustrates how age at menopause onset might impact cognition in menopausal women, and provides insight into time points to explore for the window of opportunity for hormone therapy during the menopause transition period. Hormone therapy during this critical juncture might be especially efficacious at attenuating age- and menopause- related cognitive decline, producing healthy brain aging profiles in women who retain their ovaries throughout their lifespan.

Hyperactivation of MEK1 in cortical glutamatergic neurons results in projection axon deficits and aberrant motor learning.

Abnormal extracellular signal-regulated kinase 1/2 (ERK1/2, encoded by Mapk3 and Mapk1, respectively) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates forebrain development, we conditionally expressed a hyperactive MEK1 (MAP2K1) mutant, MEK1S217/221E, in cortical excitatory neurons of mice. MEK1S217/221E expression led to persistent hyperactivation of ERK1/2 in cortical axons, but not in soma/nuclei. We noted reduced axonal arborization in multiple target domains in mutant mice and reduced the levels of the activity-dependent protein ARC. These changes did not lead to deficits in voluntary locomotion or accelerating rotarod performance. However, skilled motor learning in a single-pellet retrieval task was significantly diminished in these MEK1S217/221E mutants. Restriction of MEK1S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits but did not affect motor learning. These results suggest that cortical excitatory neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, which coincide with reduced ARC expression, and deficits in skilled motor learning. Our data indicate that neuron-autonomous decreases in long-range axonal outgrowth may be a key aspect of neuropathogenesis in RASopathies.

High and low expression of the hyperpolarization activated current (Ih ) in mouse CA1 stratum oriens interneurons.

Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological, and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking versus higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k-means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal-ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage-clamp recordings showed hyperpolarization activated current Ih was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High Ih (LTH) cells.

The PKC-ß selective inhibitor, Enzastaurin, impairs memory in middle-aged rats.

Enzastaurin is a Protein Kinase C-ß selective inhibitor that was developed to treat cancers. Protein Kinase C-ß is an important enzyme for a variety of neuronal functions; in particular, previous rodent studies have reported deficits in spatial and fear-conditioned learning and memory with lower levels of Protein Kinase C-ß. Due to Enzastaurin's mechanism of action, the present study investigated the consequences of Enzastaurin exposure on learning and memory in 12-month-old Fischer-344 male rats. Rats were treated daily with subcutaneous injections of either vehicle or Enzastaurin, and behaviorally tested using the spatial reference memory Morris Water Maze. Rats treated with Enzastaurin exhibited decreased overnight retention and poorer performance on the latter testing day, indicating a mild, but significant, memory impairment. There were no differences during the probe trial indicating that all animals were able to spatially localize the platform to the proper quadrant by the end of testing. RNA isolated from the hippocampus was analyzed using Next Generation Sequencing (Illumina). No statistically significant transcriptional differences were noted. Our findings suggest that acute Enzastaurin treatment can impair hippocampal-based learning and memory performance, with no effects on transcription in the hippocampus. We propose that care should be taken in future clinical trials that utilize Protein Kinase C-ß inhibitors, to monitor for possible cognitive effects, future research should examine if these effects are fully reversible.

Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17ß-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions.

Decreased serotonin (5-HT) function is associated with numerous cognitive and affective disorders. Women are more vulnerable to these disorders and have a lower rate of 5-HT synthesis than men. Serotonergic neurons in the dorsal raphe nucleus (DRN) are a major source of 5-HT in the forebrain and play a critical role in regulation of stress-related disorders. In particular, polymorphisms of tryptophan hydroxylase-2 (TpH2, the brain-specific, rate-limiting enzyme for 5-HT biosynthesis) are implicated in cognitive and affective disorders. Administration of 17ß-estradiol (E2), the most potent naturally circulating estrogen in women and rats, can have beneficial effects on cognitive, anxiety-like, and depressive-like behaviors. Moreover, E2 increases TpH2 mRNA in specific subregions of the DRN. Although conjugated equine estrogens (CEE) are a commonly prescribed estrogen component of hormone therapy in menopausal women, there is a marked gap in knowledge regarding how CEE affects these behaviors and the brain 5-HT system. Therefore, we compared the effects of CEE and E2 treatments on behavior and TpH2 mRNA. Female Sprague-Dawley rats were ovariectomized, administered either vehicle, CEE, or E2 and tested on a battery of cognitive, anxiety-like, and depressive-like behaviors. The brains of these animals were subsequently analyzed for TpH2 mRNA. Both CEE and E2 exerted beneficial behavioral effects, although efficacy depended on the distinct behavior and for cognition, on the task difficulty. Compared to CEE, E2 generally had more robust anxiolytic and antidepressant effects. E2 increased TpH2 mRNA in the caudal and mid DRN, corroborating previous findings. However, CEE increased TpH2 mRNA in the caudal and rostral, but not the mid, DRN, suggesting that distinct estrogens can have subregion-specific effects on TpH2 gene expression. We also found differential correlations between the level of TpH2 mRNA in specific DRN subregions and behavior, depending on the type of behavior. These distinct associations imply that cognition, anxiety-like, and depressive-like behaviors are modulated by unique serotonergic neurocircuitry, opening the possibility of novel avenues of targeted treatment for different types of cognitive and affective disorders.

Navigating to new frontiers in behavioral neuroscience: traditional neuropsychological tests predict human performance on a rodent-inspired radial-arm maze.

We constructed an 11-arm, walk-through, human radial-arm maze (HRAM) as a translational instrument to compare existing methodology in the areas of rodent and human learning and memory research. The HRAM, utilized here, serves as an intermediary test between the classic rat radial-arm maze (RAM) and standard human neuropsychological and cognitive tests. We show that the HRAM is a useful instrument to examine working memory ability, explore the relationships between rodent and human memory and cognition models, and evaluate factors that contribute to human navigational ability. One-hundred-and-fifty-seven participants were tested on the HRAM, and scores were compared to performance on a standard cognitive battery focused on episodic memory, working memory capacity, and visuospatial ability. We found that errors on the HRAM increased as working memory demand became elevated, similar to the pattern typically seen in rodents, and that for this task, performance appears similar to Miller's classic description of a processing-inclusive human working memory capacity of 7 ± 2 items. Regression analysis revealed that measures of working memory capacity and visuospatial ability accounted for a large proportion of variance in HRAM scores, while measures of episodic memory and general intelligence did not serve as significant predictors of HRAM performance. We present the HRAM as a novel instrument for measuring navigational behavior in humans, as is traditionally done in basic science studies evaluating rodent learning and memory, thus providing a useful tool to help connect and translate between human and rodent models of cognitive functioning.