Deriving early single-rosette brain organoids from human pluripotent stem cells.

Brain organoid methods are complicated by multiple rosette structures and morphological variability. We have developed a human brain organoid technique that generates self-organizing, single-rosette cortical organoids (SOSR-COs) with reproducible size and structure at early timepoints. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D developmental transition from neural plate to neural tube. Most monolayer fragments form spheres with a single central lumen. Over time, the SOSR-COs develop appropriate progenitor and cortical laminar cell types as shown by immunocytochemistry and single-cell RNA sequencing. At early time points, this method demonstrates robust structural phenotypes after chemical teratogen exposure or when modeling a genetic neurodevelopmental disorder, and should prove useful for studies of human brain development and disease modeling.

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel, CaV 1.3.

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, CaV 1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of CaV 1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of CaV 1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.

Tissue Plasminogen Activator in Central Nervous System Physiology and Pathology: From Synaptic Plasticity to Alzheimer's Disease.

Tissue plasminogen activator's (tPA) fibrinolytic function in the vasculature is well-established. This specific role for tPA in the vasculature, however, contrasts with its pleiotropic activities in the central nervous system. Numerous physiological and pathological functions have been attributed to tPA in the central nervous system, including neurite outgrowth and regeneration; synaptic and spine plasticity; neurovascular coupling; neurodegeneration; microglial activation; and blood-brain barrier permeability. In addition, multiple substrates, both plasminogen-dependent and -independent, have been proposed to be responsible for tPA's action(s) in the central nervous system. This review aims to dissect a subset of these different functions and the different molecular mechanisms attributed to tPA in the context of learning and memory. We start from the original research that identified tPA as an immediate-early gene with a putative role in synaptic plasticity to what is currently known about tPA's role in a learning and memory disorder, Alzheimer's disease. We specifically focus on studies demonstrating tPA's involvement in the clearance of amyloid-ß and neurovascular coupling. In addition, given that tPA has been shown to regulate blood-brain barrier permeability, which is perturbed in Alzheimer's disease, this review also discusses tPA-mediated vascular dysfunction and possible alternative mechanisms of action for tPA in Alzheimer's disease pathology.

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

Modeling UBQLN2-mediated neurodegenerative disease in mice: Shared and divergent properties of wild type and mutant UBQLN2 in phase separation, subcellular localization, altered proteostasis pathways, and selective cytotoxicity.

The ubiquitin-binding proteasomal shuttle protein UBQLN2 is implicated in common neurodegenerative disorders due to its accumulation in disease-specific aggregates and, when mutated, directly causes familial frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). Like other proteins linked to FTD/ALS, UBQLN2 undergoes phase separation to form condensates. The relationship of UBQLN2 phase separation and accumulation to neurodegeneration, however, remains uncertain. Employing biochemical, neuropathological and behavioral assays, we studied the impact of overexpressing WT or mutant UBQLN2 in the CNS of transgenic mice. Expression of UBQLN2 harboring a pathogenic mutation (P506T) elicited profound and widespread intraneuronal inclusion formation and aggregation without prominent neurodegenerative or behavioral changes. Both WT and mutant UBQLN2 formed ubiquitin- and P62-positive inclusions in neurons, supporting the view that UBQLN2 is intrinsically prone to phase separate, with the size, shape and frequency of inclusions depending on expression level and the presence or absence of a pathogenic mutation. Overexpression of WT or mutant UBQLN2 resulted in a dose-dependent decrease in levels of a key interacting chaperone, HSP70, as well as dose-dependent profound degeneration of the retina. We conclude that, at least in mice, robust aggregation of a pathogenic form of UBQLN2 is insufficient to cause neuronal loss recapitulating that of human FTD/ALS. Our results nevertheless support the view that altering the normal cellular balance of UBQLN2, whether wild type or mutant protein, has deleterious effects on cells of the CNS and retina that likely reflect perturbations in ubiquitin-dependent protein homeostasis.

Turning strains into strengths for understanding psychiatric disorders.

There is a paucity in the development of new mechanistic insights and therapeutic approaches for treating psychiatric disease. One of the major challenges is reflected in the growing consensus that risk for these diseases is not determined by a single gene, but rather is polygenic, arising from the action and interaction of multiple genes. Canonically, experimental models in mice have been designed to ascertain the relative contribution of a single gene to a disease by systematic manipulation (e.g., mutation or deletion) of a known candidate gene. Because these studies have been largely carried out using inbred isogenic mouse strains, in which there is no (or very little) genetic diversity among subjects, it is difficult to identify unique allelic variants, gene modifiers, and epigenetic factors that strongly affect the nature and severity of these diseases. Here, we review various methods that take advantage of existing genetic diversity or that increase genetic variance in mouse models to (1) strengthen conclusions of single-gene function; (2) model diversity among human populations; and (3) dissect complex phenotypes that arise from the actions of multiple genes.

The role of L-type calcium channels in neuronal excitability and aging.

Over the last two decades there has been significant progress towards understanding the neural substrates that underlie age-related cognitive decline. Although many of the exact molecular and cellular mechanisms have yet to be fully understood, there is consensus that alterations in neuronal calcium homeostasis contribute to age-related deficits in learning and memory. Furthermore, it is thought that the age-related changes in calcium homeostasis are driven, at least in part, by changes in calcium channel expression. In this review, we focus on the role of a specific class of calcium channels: L-type voltage-gated calcium channels (LVGCCs). We provide the reader with a general introduction to voltage-gated calcium channels, followed by a more detailed description of LVGCCs and how they serve to regulate neuronal excitability via the post burst afterhyperpolarization (AHP). We conclude by reviewing studies that link the slow component of the AHP to learning and memory, and discuss how age-related increases in LVGCC expression may underlie cognitive decline by mediating a decrease in neuronal excitability.

Altered Synaptic Drive onto Birthdated Dentate Granule Cells in Experimental Temporal Lobe Epilepsy.

Dysregulated adult hippocampal neurogenesis occurs in many temporal lobe epilepsy (TLE) models. Most dentate granule cells (DGCs) generated in response to an epileptic insult develop features that promote increased excitability, including ectopic location, persistent hilar basal dendrites (HBDs), and mossy fiber sprouting. However, some appear to integrate normally and even exhibit reduced excitability compared to other DGCs. To examine the relationship between DGC birthdate, morphology, and network integration in a model of TLE, we retrovirally birthdated either early-born [EB; postnatal day (P)7] or adult-born (AB; P60) DGCs. Male rats underwent pilocarpine-induced status epilepticus (SE) or sham treatment at P56. Three to six months after SE or sham treatment, we used whole-cell patch-clamp and fluorescence microscopy to record spontaneous excitatory and inhibitory currents from birthdated DGCs. We found that both AB and EB populations of DGCs recorded from epileptic rats received increased excitatory input compared with age-matched controls. Interestingly, when AB populations were separated into normally integrated (normotopic) and aberrant (ectopic or HBD-containing) subpopulations, only the aberrant populations exhibited a relative increase in excitatory input (amplitude, frequency, and charge transfer). The ratio of excitatory-to-inhibitory input was most dramatically upregulated for ectopically localized DGCs. These data provide definitive physiological evidence that aberrant integration of post-SE, AB DGCs contributes to increased synaptic drive and support the idea that ectopic DGCs serve as putative hub cells to promote seizures.SIGNIFICANCE STATEMENT Adult dentate granule cell (DGC) neurogenesis is altered in rodent models of temporal lobe epilepsy (TLE). Some of the new neurons show abnormal morphology and integration, but whether adult-generated DGCs contribute to the development of epilepsy is controversial. We examined the synaptic inputs of age-defined populations of DGCs using electrophysiological recordings and fluorescent retroviral reporter birthdating. DGCs generated neonatally were compared with those generated in adulthood, and adult-born (AB) neurons with normal versus aberrant morphology or integration were examined. We found that AB, ectopically located DGCs exhibit the most pro-excitatory physiological changes, implicating this population in seizure generation or progression.

Environmental variables that ameliorate extinction learning deficits in the 129S1/SvlmJ mouse strain.

Fear conditioning is an associative learning process by which organisms learn to avoid environmental stimuli that are predictive of aversive outcomes. Fear extinction learning is a process by which avoidance of fear-conditioned stimuli is attenuated when the environmental stimuli is no longer predictive of the aversive outcome. Aberrant fear conditioning and extinction learning are key elements in the development of several anxiety disorders. The 129S1 inbred strain of mice is used as an animal model for maladaptive fear learning because this strain has been shown to generalize fear to other nonaversive stimuli and is less capable of extinguishing fear responses relative to other mouse strains, such as the C57BL/6. Here we report new environmental manipulations that enhance fear and extinction learning, including the ability to discriminate between an aversively paired tone and a neutral tone, in both the 129S1 and C57BL/6 strains of mice. Specifically, we show that discontinuous ("pipped") tone stimuli significantly enhance within-session extinction learning and the discrimination between neutral and aversively paired stimuli in both strains. Furthermore, we find that extinction training in novel contexts significantly enhances the consolidation and recall of extinction learning for both strains. Cumulatively, these results underscore how environmental changes can be leveraged to ameliorate maladaptive learning in animal models and may advance cognitive and behavioral therapeutic strategies.

A novel mouse model of the aged brain: Over-expression of the L-type voltage-gated calcium channel CaV1.3.

The aged population is growing rapidly, which has sparked tremendous interest in elucidating mechanisms of aging in both the body and the brain. Animal models have become an indispensable tool in biomedical science, but because of the cost and extended timeframe associated with aging animals to appropriate time points, studies that rely on using aged animals are often not feasible. Somewhat surprisingly, there are relatively few animal models that have been specifically engineered to mimic physiological changes known to occur during "normal" aging. Developing transgenic animal models that faithfully mimic key aspects of aging would likely be of great utility in studying both age-related deficits in the absence of overt pathology as well as an adjunct for transgenic models of diseases where aging is a primary risk factor. In particular, there are several alterations in the aged brain that are amenable to being modeled genetically. We have focused on one key aspect that has been repeatedly demonstrated in aged animals - an increase in the L-type voltage-gated calcium channel CaV1.3. Here we present a novel transgenic mouse line in which expression of CaV1.3 is increased by approximately 50% in the forebrain of young mice. These mice do not display any overt physical or non-cognitive deficits, exhibiting normal exploratory behavior, motor function, and affective-like responses, suggesting that these mice can be successfully deployed to assess the impact of an "aged brain" in a variety of conditions.

Conversion of short-term to long-term memory in the novel object recognition paradigm.

It is well-known that stress can significantly impact learning; however, whether this effect facilitates or impairs the resultant memory depends on the characteristics of the stressor. Investigation of these dynamics can be confounded by the role of the stressor in motivating performance in a task. Positing a cohesive model of the effect of stress on learning and memory necessitates elucidating the consequences of stressful stimuli independently from task-specific functions. Therefore, the goal of this study was to examine the effect of manipulating a task-independent stressor (elevated light level) on short-term and long-term memory in the novel object recognition paradigm. Short-term memory was elicited in both low light and high light conditions, but long-term memory specifically required high light conditions during the acquisition phase (familiarization trial) and was independent of the light level during retrieval (test trial). Additionally, long-term memory appeared to be independent of stress-mediated glucocorticoid release, as both low and high light produced similar levels of plasma corticosterone, which further did not correlate with subsequent memory performance. Finally, both short-term and long-term memory showed no savings between repeated experiments suggesting that this novel object recognition paradigm may be useful for longitudinal studies, particularly when investigating treatments to stabilize or enhance weak memories in neurodegenerative diseases or during age-related cognitive decline.

Hippocampal pyramidal neurons comprise two distinct cell types that are countermodulated by metabotropic receptors.

Relating the function of neuronal cell types to information processing and behavior is a central goal of neuroscience. In the hippocampus, pyramidal cells in CA1 and the subiculum process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information, which they transmit throughout the brain. Do these cells constitute a single class or are there multiple cell types with specialized functions? Using unbiased cluster analysis, we show that there are two morphologically and electrophysiologically distinct principal cell types that carry hippocampal output. We show further that these two cell types are inversely modulated by the synergistic action of glutamate and acetylcholine acting on metabotropic receptors that are central to hippocampal function. Combined with prior connectivity studies, our results support a model of hippocampal processing in which the two pyramidal cell types are predominantly segregated into two parallel pathways that process distinct modalities of information.

Of mice and intrinsic excitability: genetic background affects the size of the postburst afterhyperpolarization in CA1 pyramidal neurons.

As the use of genetically engineered mice has become increasingly prevalent in neurobiological research, evidence has steadily accumulated that substantial differences exist between strains. Although a number of studies have reported effects of genetic background on behavior, few have focused on differences in neurophysiology. The postburst afterhyperpolarization (AHP) is an important determinant of intrinsic neuronal excitability and has been suggested to play a critical role in the cellular mechanisms underlying learning and memory. Using whole cell current-clamp recordings of CA1 pyramidal neurons, we examined the magnitude of different phases of the AHP (peak, medium, and slow) in two commonly used genetic backgrounds, C57BL/6 (B6) and 129SvEv (129), as well as in an F2 hybrid B6:129 background (F2). We found that neurons from B6 and F2 animals exhibited a significantly larger AHP compared with 129 animals and that this difference was consistent across all phases. Furthermore, our recordings revealed a marked dichotomy in the shape of the AHP waveform, which was independent of genetic background. Approximately 60% of cells exhibited an AHP with a sharp transition between the peak AHP and medium AHP, whereas the remaining 40% exhibited a more gradual transition. Our data add to the growing body of work suggesting that genetic background can affect neuronal function as well as behavior. In addition, these results highlight the innate heterogeneity of CA1 pyramidal neurons, even within a single genetic background. These differences should be taken into consideration during the analysis and comparison of experimental results.

Plasticity of burst firing induced by synergistic activation of metabotropic glutamate and acetylcholine receptors.

Subiculum, the primary efferent pathway of hippocampus, participates in memory for spatial tasks, relapse to drug abuse, and temporal lobe seizures. Subicular pyramidal neurons exhibit low-threshold burst firing driven by a spike afterdepolarization. Here we report that burst firing can be regulated by stimulation of afferent projections to subiculum. Unlike synaptic plasticity, burst plasticity did not require synaptic depolarization, activation of AMPA or NMDA receptors, or action potential firing. Rather, enhancement of burst firing required synergistic activation of group I, subtype 1 metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChR). When either of these receptors was blocked, a suppression of bursting was revealed, which in turn was blocked by antagonists of group I, subtype 5 mGluRs. These results indicate that the output of subiculum can be strongly and bidirectionally regulated by activation of glutamatergic inputs within the hippocampus and cholinergic afferents from the medial septum.

Psychostimulant-induced plasticity of intrinsic neuronal excitability in ventral subiculum.

Psychostimulant drugs such as amphetamine are prescribed to increase vigilance, suppress appetite, and treat attention disorders, but they powerfully activate the dopamine system and have serious abuse potential. Repeated psychostimulant exposure induces neuronal plasticity within the mesolimbic dopamine system. Here we present evidence that repeated amphetamine exposure results in a suppression of intrinsic neuronal excitability in the ventral subiculum, a hippocampal region that activates dopamine neurotransmission. We used patch-clamp recordings from brain slices obtained at different times after withdrawal from repeated amphetamine exposure to determine the long-term effects of amphetamine on subicular excitability. Using several postsynaptic indices of sodium channel function, our results show that excitability is decreased for days, but not weeks, after repeated amphetamine exposure. The resulting increase in action potential threshold and decrease in postsynaptic amplification of excitatory synaptic input provide the first direct evidence that psychostimulants induce plasticity of hippocampal output and suggest one mechanism by which drug withdrawal may influence limbic dopamine-dependent learning and memory.