Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging.

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.

Multiline Orthogonal Scanning Temporal Focusing (mosTF) Microscopy for Scattering Reduction in High-speed in vivo Brain Imaging.

Temporal focusing two-photon microscopy enables high resolution imaging of fine structures in vivo over a large volume. A limitation of temporal focusing is that signal-to-background ratio and resolution degrade rapidly with increasing imaging depth. This degradation originates from the scattered emission photons are widely distributed resulting in a strong background. We have developed Multiline Orthogonal Scanning Temporal Focusing (mosTF) microscopy that overcomes this problem. mosTF captures a sequence of images at each scan location of the excitation line, followed by a reconstruction algorithm reassigns scattered photons back to the correct scan position. We demonstrate mosTF by acquiring mice neuronal images in vivo. Our results show remarkably improvements with mosTF for in vivo brain imaging while maintaining its speed advantage.

De-scattering Deep Neural Network Enables Fast Imaging of Spines through Scattering Media by Temporal Focusing Microscopy.

Today the gold standard for in vivo imaging through scattering tissue is point-scanning two-photon microscopy (PSTPM). Especially in neuroscience, PSTPM is widely used for deep-tissue imaging in the brain. However, due to sequential scanning, PSTPM is slow. Temporal focusing microscopy (TFM), on the other hand, focuses femtosecond pulsed laser light temporally while keeping wide-field illumination, and is consequently much faster. However, due to the use of a camera detector, TFM suffers from the scattering of emission photons. As a result, TFM produces images of poor quality, obscuring fluorescent signals from small structures such as dendritic spines. In this work, we present a de-scattering deep neural network (DeScatterNet) to improve the quality of TFM images. Using a 3D convolutional neural network (CNN) we build a map from TFM to PSTPM modalities, to enable fast TFM imaging while maintaining high image quality through scattering media. We demonstrate this approach for in vivo imaging of dendritic spines on pyramidal neurons in the mouse visual cortex. We quantitatively show that our trained network rapidly outputs images that recover biologically relevant features previously buried in the scattered fluorescence in the TFM images. In vivo imaging that combines TFM and the proposed neural network is one to two orders of magnitude faster than PSTPM but retains the high quality necessary to analyze small fluorescent structures. The proposed approach could also be beneficial for improving the performance of many speed-demanding deep-tissue imaging applications, such as in vivo voltage imaging.

De-scattering with Excitation Patterning enables rapid wide-field imaging through scattering media.

Nonlinear optical microscopy has enabled in vivo deep tissue imaging on the millimeter scale. A key unmet challenge is its limited throughput especially compared to rapid wide-field modalities that are used ubiquitously in thin specimens. Wide-field imaging methods in tissue specimens have found successes in optically cleared tissues and at shallower depths, but the scattering of emission photons in thick turbid samples severely degrades image quality at the camera. To address this challenge, we introduce a novel technique called De-scattering with Excitation Patterning or "DEEP," which uses patterned nonlinear excitation followed by computational imaging-assisted wide-field detection. Multiphoton temporal focusing allows high-resolution excitation patterns to be projected deep inside specimen at multiple scattering lengths due to the use of long wavelength light. Computational reconstruction allows high-resolution structural features to be reconstructed from tens to hundreds of DEEP images instead of millions of point-scanning measurements.

Scanless volumetric imaging by selective access multifocal multiphoton microscopy.

Simultaneous, high-resolution imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of submicrometer-sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume. We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites.

Scattering reduction by structured light illumination in line-scanning temporal focusing microscopy.

Line-scanning temporal focusing microscopy (LineTFM) is capable of imaging biological samples more than 10 times faster than two-photon laser point-scanning microscopy (TPLSM), while achieving nearly the same lateral and axial spatial resolution. However, the image contrast taken by LineTFM is lower than that by TPLSM because LineTFM is severely influenced by biological tissue scattering. To reject the scattered photons, we implemented LineTFM using both structured illumination and uniform illumination combined with the HiLo post-processing algorithm, called HiLL microscopy (HiLo-Line-scanning temporal focusing microscopy). HiLL microscopy significantly reduces tissue scattering and improves image contrast. We demonstrate HiLL microscopy with in vivo brain imaging. This approach could potentially find applications in monitoring fast dynamic events and in mapping high resolution structures over a large volume.

Functional implications of inhibitory synapse placement on signal processing in pyramidal neuron dendrites.

A rich literature describes inhibitory innervation of pyramidal neurons in terms of the distinct inhibitory cell types that target the soma, axon initial segment, or dendritic arbor. Less attention has been devoted to how localization of inhibition to specific parts of the pyramidal dendritic arbor influences dendritic signal detection and integration. The effect of inhibitory inputs can vary based on their placement on dendritic spines versus shaft, their distance from the soma, and the branch order of the dendrite they inhabit. Inhibitory synapses are also structurally dynamic, and the implications of these dynamics depend on their dendritic location. Here we consider the heterogeneous roles of inhibitory synapses as defined by their strategic placement on the pyramidal cell dendritic arbor.

Adolescent pruning and stabilization of dendritic spines on cortical layer 5 pyramidal neurons do not depend on gonadal hormones.

Pyramidal neurons in the neocortex receive a majority of their synapses on dendritic spines, whose growth, gain, and loss regulate the strength and identity of neural connections. Juvenile brains typically show higher spine density and turnover compared to adult brains, potentially enabling greater capacity for experience-dependent circuit 'rewiring'. Although spine pruning and stabilization in frontal cortex overlap with pubertal milestones, it is unclear if gonadal hormones drive these processes. To address this question, we used hormone manipulations and in vivo 2-photon microscopy to test for a causal relationship between pubertal hormones and spine pruning and stabilization in layer 5 neurons in the frontal cortex of female mice. We found that spine density, gains, and losses decreased from P27 to P60 and that these measures were not affected by pre-pubertal hormone injections or ovariectomy. Further analyses of spine morphology after manipulation of gonadal hormones suggest that gonadal hormones may play a role in morphological maturation and dynamics. Our data help to segregate hormone-sensitive and hormone-insensitive maturational processes that occur simultaneously in dorsomedial frontal cortex. These data provide more specific insight into adolescent development and may have implications for understanding the neurodevelopmental effects of changes in pubertal timing in humans.

Age, sex, and gonadal hormones differently influence anxiety- and depression-related behavior during puberty in mice.

Anxiety and depression symptoms increase dramatically during adolescence, with girls showing a steeper increase than boys after puberty onset. The timing of the onset of this sex bias led us to hypothesize that ovarian hormones contribute to depression and anxiety during puberty. In humans, it is difficult to disentangle direct effects of gonadal hormones from social and environmental factors that interact with pubertal development to influence mental health. To test the role of gonadal hormones in anxiety- and depression-related behavior during puberty, we manipulated gonadal hormones in mice while controlling social and environmental factors. Similar to humans, we find that mice show an increase in depression-related behavior from pre-pubertal to late-pubertal ages, but this increase is not dependent on gonadal hormones and does not differ between sexes. Anxiety-related behavior, however, is more complex during puberty, with differences that depend on sex, age, behavioral test, and hormonal status. Briefly, males castrated before puberty show greater anxiety-related behavior during late puberty compared to intact males, while pubertal females are unaffected by ovariectomy or hormone injections in all assays except the marble burying test. Despite this sex-specific effect of pubertal hormones on anxiety-related behavior, we find no sex differences in intact young adults, suggesting that males and females use separate mechanisms to converge on a similar behavioral phenotype. Our results are consistent with anxiolytic effects of testicular hormones during puberty in males but are not consistent with a causal role for ovarian hormones in increasing anxiety- and depression-related behavior during puberty in females.

Ovarian Hormones Organize the Maturation of Inhibitory Neurotransmission in the Frontal Cortex at Puberty Onset in Female Mice.

The frontal cortex matures late in development, showing dramatic changes after puberty onset, yet few experiments have directly tested the role of pubertal hormones in cortical maturation. One mechanism thought to play a primary role in regulating the maturation of the neocortex is an increase in inhibitory neurotransmission, which alters the balance of excitation and inhibition. We hypothesized that pubertal hormones could regulate maturation of the frontal cortex by this mechanism. Here, we report that manipulations of gonadal hormones do significantly alter the maturation of inhibitory neurotransmission in the cingulate region of the mouse medial frontal cortex, an associative region that matures during the pubertal transition and is implicated in decision making, learning, and psychopathology. We find that inhibitory neurotransmission, but not excitatory neurotransmission, increases onto cingulate pyramidal neurons during peri-pubertal development and that this increase can be blocked by pre-pubertal, but not post-pubertal, gonadectomy. We next used pre-pubertal hormone treatment to model early puberty onset, a phenomenon increasingly observed in girls living in developed nations. We find that pre-pubertal hormone treatment drives an early increase in inhibitory neurotransmission in the frontal cortex, but not the somatosensory cortex, suggesting that earlier puberty can advance cortical maturation in a regionally specific manner. Pre-pubertal hormone treatment also accelerates maturation of tonic inhibition and performance in a frontal-cortex-dependent reversal-learning task. These data provide rare evidence of enduring, organizational effects of ovarian hormones at puberty and provide a potential mechanism by which gonadal hormones could regulate the maturation of the associative neocortex.

Soluble oligomeric amyloid-ß induces calcium dyshomeostasis that precedes synapse loss in the living mouse brain.

BACKGROUND: Amyloid-ß oligomers (oAß) are thought to mediate neurotoxicity in Alzheimer's disease (AD), and previous studies in AD transgenic mice suggest that calcium dysregulation may contribute to these pathological effects. Even though AD mouse models remain a valuable resource to investigate amyloid neurotoxicity, the concomitant presence of soluble Aß species, fibrillar Aß, and fragments of amyloid precursor protein (APP) complicate the interpretation of the phenotypes. METHOD: To explore the specific contribution of soluble oligomeric Aß (oAß) to calcium dyshomeostasis and synaptic morphological changes, we acutely exposed the healthy mouse brain, at 3 to 6 months of age, to naturally occurring soluble oligomers and investigated their effect on calcium levels using in vivo multiphoton imaging. RESULTS: We observed a dramatic increase in the levels of neuronal resting calcium, which was dependent upon extracellular calcium influx and activation of NMDA receptors. Ryanodine receptors, previously implicated in AD models, did not appear to be primarily involved using this experimental setting. We used the high resolution cortical volumes acquired in-vivo to measure the effect on synaptic densities and observed that, while spine density remained stable within the first hour of oAß exposure, a significant decrease in the number of dendritic spines was observed 24 h post treatment, despite restoration of intraneuronal calcium levels at this time point. CONCLUSIONS: These observations demonstrate a specific effect of oAß on NMDA-mediated calcium influx, which triggers synaptic collapse in vivo. Moreover, this work leverages a method to quantitatively measure calcium concentration at the level of neuronal processes, cell bodies and single synaptic elements repeatedly and thus can be applicable to testing putative drugs and/or other intervention methodologies.

Does puberty mark a transition in sensitive periods for plasticity in the associative neocortex?

Postnatal brain development is studded with sensitive periods during which experience dependent plasticity is enhanced. This enables rapid learning from environmental inputs and reorganization of cortical circuits that matches behavior with environmental contingencies. Significant headway has been achieved in characterizing and understanding sensitive period biology in primary sensory cortices, but relatively little is known about sensitive period biology in associative neocortex. One possible mediator is the onset of puberty, which marks the transition to adolescence, when animals shift their behavior toward gaining independence and exploring their social world. Puberty onset correlates with reduced behavioral plasticity in some domains and enhanced plasticity in others, and therefore may drive the transition from juvenile to adolescent brain function. Pubertal onset is also occurring earlier in developed nations, particularly in unserved populations, and earlier puberty is associated with vulnerability for substance use, depression and anxiety. In the present article we review the evidence that supports a causal role for puberty in developmental changes in the function and neurobiology of the associative neocortex. We also propose a model for how pubertal hormones may regulate sensitive period plasticity in associative neocortex. We conclude that the evidence suggests puberty onset may play a causal role in some aspects of associative neocortical development, but that further research that manipulates puberty and measures gonadal hormones is required. We argue that further work of this kind is urgently needed to determine how earlier puberty may negatively impact human health and learning potential. This article is part of a Special Issue entitled SI: Adolescent plasticity.

Brief cognitive training interventions in young adulthood promote long-term resilience to drug-seeking behavior.

Environmental stress and deprivation increase vulnerability to substance use disorders in humans and promote drug-seeking behavior in animal models. In contrast, experiences of mastery and stability may shape neural circuitry in ways that build resilience to future challenges. Cognitive training offers a potential intervention for reducing vulnerability in the face of environmental stress or deprivation. Here, we test the hypothesis that brief cognitive training can promote long-term resilience to one measure of drug-seeking behavior, cocaine conditioned place preference (CPP), in mice. In young adulthood, mice underwent cognitive training, received rewards while exploring a training arena (i.e. yoked control), or remained in their home cages. Beginning 4 weeks after cessation of training, we conditioned mice in a CPP paradigm and then tested them weekly for CPP maintenance or daily for CPP extinction. We found that a brief 9-day cognitive training protocol reduced maintenance of cocaine CPP when compared to standard housed and yoked conditions. This beneficial effect persisted long after cessation of the training, as mice remained in their home cages for 4 weeks between training and cocaine exposure. When mice were tested for CPP on a daily extinction schedule, we found that all trained and yoked groups that left their home cages to receive rewards in a training arena showed significant extinction of CPP, while mice kept in standard housing for the same period did not extinguish CPP. These data suggest that in early adulthood, deprivation may confer vulnerability to drug-seeking behavior and that brief interventions may promote long-term resilience.

Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens.

The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.

A causal link between prediction errors, dopamine neurons and learning.

Situations in which rewards are unexpectedly obtained or withheld represent opportunities for new learning. Often, this learning includes identifying cues that predict reward availability. Unexpected rewards strongly activate midbrain dopamine neurons. This phasic signal is proposed to support learning about antecedent cues by signaling discrepancies between actual and expected outcomes, termed a reward prediction error. However, it is unknown whether dopamine neuron prediction error signaling and cue-reward learning are causally linked. To test this hypothesis, we manipulated dopamine neuron activity in rats in two behavioral procedures, associative blocking and extinction, that illustrate the essential function of prediction errors in learning. We observed that optogenetic activation of dopamine neurons concurrent with reward delivery, mimicking a prediction error, was sufficient to cause long-lasting increases in cue-elicited reward-seeking behavior. Our findings establish a causal role for temporally precise dopamine neuron signaling in cue-reward learning, bridging a critical gap between experimental evidence and influential theoretical frameworks.