In Vivo Mesoscopic Voltage-Sensitive Dye Imaging of Brain Activation.
ABSTRACT: Functional mapping of brain activity is important in elucidating how neural networks operate in the living brain. The whisker sensory system of rodents is an excellent model to study peripherally evoked neural activity in the central nervous system. Each facial whisker is represented by discrete modules of neurons all along the pathway leading to the neocortex. These modules are called "barrels" in layer 4 of the primary somatosensory cortex. Their location (approximately 300-500 μm below cortical surface) allows for convenient imaging of whisker-evoked neural activity in vivo. Fluorescence laminar optical tomography (FLOT) provides depth-resolved fluorescence molecular information with an imaging depth of a few millimeters. Angled illumination and detection configurations can improve both resolution and penetration depth. We applied angled FLOT (aFLOT) to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. A 100 μm capillary and a pair of microelectrodes were inserted to the mouse brain to test the capability of the imaging system. The results show that it is possible to obtain 3D functional maps of the sensory periphery in the brain. This approach can be broadly applicable to functional imaging of other brain structures.
Project description:The whisker system of rodents is an excellent model to study peripherally evoked neural activity in the brain. Discrete neural modules represent each whisker in the somatosensory cortex ("barrels"), thalamus ("barreloids"), and brain stem ("barrelettes"). Stimulation of a single whisker evokes neural activity sequentially in its corresponding barrelette, barreloid, and barrel. Conventional optical imaging of functional activation in the brain is limited to surface structures such as the cerebral cortex. To access subcortical structures and image sensory-evoked neural activity, we designed a needle-based optical system using gradient-index (GRIN) rod lens. We performed voltage-sensitive dye imaging (VSDi) with GRIN rod lens to visualize neural activity evoked in the thalamic barreloids by deflection of whiskers in vivo. We stimulated several whiskers together to determine the sensitivity of our approach in differentiating between different barreloid responses. We also carried out stimulation of different whiskers at different times. Finally, we used muscimol in the barrel cortex to silence the corticothalamic inputs while imaging in the thalamus. Our results show that it is possible to obtain functional maps of the sensory periphery in deep brain structures such as the thalamic barreloids. Our approach can be broadly applicable to functional imaging of other core brain structures.
Project description:The whisker system is an important sensory organ with extensive neural representations in the brain of the mouse. Patterned neural modules (barrelettes) in the ipsilateral principal sensory nucleus of the trigeminal nerve (PrV) correspond to the whiskers. Axons of the PrV barrelette neurons cross the midline and confer the whisker-related patterning to the contralateral ventroposteromedial nucleus of the thalamus, and subsequently to the cortex. In this way, specific neural modules called barreloids and barrels in the contralateral thalamus and cortex represent each whisker. Partial midline crossing of the PrV axons, in a conditional Robo3 mutant (Robo3R3-5cKO) mouse line, leads to the formation of bilateral whisker maps in the ventroposteromedial, as well as the barrel cortex. We used voltage-sensitive dye optical imaging and somatosensory and motor behavioral tests to characterize the consequences of bifacial maps in the thalamocortical system. Voltage-sensitive dye optical imaging verified functional, bilateral whisker representation in the barrel cortex and activation of distinct cortical loci following ipsilateral and contralateral stimulation of the specific whiskers. The mutant animals were comparable with the control animals in sensorimotor tests. However, they showed noticeable deficits in all of the whisker-dependent or -related tests, including Y-maze exploration, horizontal surface approach, bridge crossing, gap crossing, texture discrimination, floating in water, and whisking laterality. Our results indicate that bifacial maps along the thalamocortical system do not offer a functional advantage. Instead, they lead to impairments, possibly due to the smaller size of the whisker-related modules and interference between the ipsilateral and contralateral whisker representations in the same thalamus and cortex.SIGNIFICANCE STATEMENT The whisker sensory system plays a quintessentially important role in exploratory behavior of mice and other nocturnal rodents. Here, we studied a novel mutant mouse line, in which the projections from the brainstem to the thalamus are disrupted. This led to formation of bilateral whisker maps in both the thalamus and the cortex. The two whisker maps crowd in a space normally devoted to the contralateral map alone and in a nonoverlapping fashion. Stimulation of the whiskers on either side activates the corresponding region of the map. Mice with bilateral whisker maps perform well in general sensorimotor tasks but show poor performance in specific tests that require whisker-dependent tactile discrimination. These observations indicate that contralateral, instead of bilateral, representation of the sensory space plays a critical role in acuity and fine discrimination during somesthesis.
Project description:Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization, and quantification of the activity of all neurons across the entire brain, which has not, to date, been achieved in the mammalian brain. We introduce a pipeline for high-speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Last, we combine activity mapping with axon tracing to uncover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available.
Project description:Invariant sensory coding is the robust coding of some sensory information (e.g., stimulus type) despite major changes in other sensory parameters (e.g., stimulus strength). The contribution of large populations of neurons (ensembles) to invariant sensory coding is not well understood, but could offer distinct advantages over invariance in single cell receptive fields. To test invariant sensory coding in neuronal ensembles evoked by single whisker stimulation as early as primary sensory cortex, we recorded detailed spatiotemporal movies of evoked ensemble activity through the depth of rat barrel cortex using microelectrode arrays. We found that an emergent property of whisker evoked ensemble activity, its spatiotemporal profile, was notably invariant across major changes in stimulus amplitude (up to >200-fold). Such ensemble-based invariance was found for single whisker stimulation as well as for the integrated profile of activity evoked by the more naturalistic stimulation of the entire whisker array. Further, the integrated profile of whisker array evoked ensemble activity and its invariance to stimulus amplitude shares striking similarities to "funneled" tactile perception in humans. We therefore suggest that ensemble-based invariance could provide a robust neurobiological substrate for invariant sensory coding and integration at an early stage of cortical sensory processing already in primary sensory cortex.
Project description:Absorption or fluorescence-based two-dimensional (2-D) optical imaging is widely employed in functional brain imaging. The image is a weighted sum of the real signal from the tissue at different depths. This weighting function is defined as "depth sensitivity." Characterizing depth sensitivity and spatial resolution is important to better interpret the functional imaging data. However, due to light scattering and absorption in biological tissues, our knowledge of these is incomplete. We use Monte Carlo simulations to carry out a systematic study of spatial resolution and depth sensitivity for 2-D optical imaging methods with configurations typically encountered in functional brain imaging. We found the following: (i) the spatial resolution is <200 μm for NA≤0.2 or focal plane depth≤300 μm. (ii) More than 97% of the signal comes from the top 500 μm of the tissue. (iii) For activated columns with lateral size larger than spatial resolution, changing numerical aperature (NA) and focal plane depth does not affect depth sensitivity. (iv) For either smaller columns or large columns covered by surface vessels, increasing NA and/or focal plane depth may improve depth sensitivity at deeper layers. Our results provide valuable guidance for the optimization of optical imaging systems and data interpretation.
Project description:Alzheimer's disease (AD) is the most common form of dementia. One of the neuropathological hallmarks of AD is the accumulation of amyloid-? plaques. Overexpression of human amyloid precursor protein in transgenic mice induces hippocampal and neocortical amyloid-? accumulation and plaque deposition that increases with age. The impact of these effects on neuronal population responses and network activity in sensory cortex is not well understood. We used Voltage Sensitive Dye Imaging, to investigate at high spatial and temporal resolution, the sensory evoked population responses in the barrel cortex of aged transgenic (Tg) mice and of age-matched non-transgenic littermate controls (Ctrl) mice. We found that a whisker deflection evoked abnormal sensory responses in the barrel cortex of Tg mice. The response amplitude and the spatial spread of the cortical responses were significantly larger in Tg than in Ctrl mice. At the network level, spontaneous activity was less synchronized over cortical space than in Ctrl mice, however synchronization during evoked responses induced by whisker deflection did not differ between the two groups. Thus, the presence of elevated A? and plaques may alter population responses and disrupts neural synchronization in large-scale networks, leading to abnormalities in sensory processing.
Project description:The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here, we report on a novel volume imaging and 3D tracking technique that monitors whole brain neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional imaging of neural activity during visually evoked and prey capture behaviors in larval zebrafish.
Project description:How homeostatic processes contribute to map plasticity and stability in sensory cortex is not well-understood. Classically, sensory deprivation first drives rapid Hebbian weakening of spiking responses to deprived inputs, which is followed days later by a slow homeostatic increase in spiking responses mediated by excitatory synaptic scaling. Recently, more rapid homeostasis by inhibitory circuit plasticity has been discovered in visual cortex, but whether this process occurs in other brain areas is not known. We tested for rapid homeostasis in layer 2/3 (L2/3) of rodent somatosensory cortex, where D-row whisker deprivation drives Hebbian weakening of whisker-evoked spiking responses after an unexplained initial delay, but no homeostasis of deprived whisker responses is known. We hypothesized that the delay reflects rapid homeostasis through disinhibition, which masks the onset of Hebbian weakening of L2/3 excitatory input. We found that deprivation (3 d) transiently increased whisker-evoked spiking responses in L2/3 single units before classical Hebbian weakening (?5 d), whereas whisker-evoked synaptic input was reduced during both periods. This finding suggests a transient homeostatic increase in L2/3 excitability. In whole-cell recordings from L2/3 neurons in vivo, brief deprivation decreased whisker-evoked inhibition more than excitation and increased the excitation-inhibition ratio. In contrast, synaptic scaling and increased intrinsic excitability were absent. Thus, disinhibition is a rapid homeostatic plasticity mechanism in rodent somatosensory cortex that transiently maintains whisker-evoked spiking in L2/3, despite the onset of Hebbian weakening of excitatory input.
Project description:The place theory proposed by Jeffress (1948) is still the dominant model of how the brain represents the movement of sensory stimuli between sensory receptors. According to the place theory, delays in signalling between neurons, dependent on the distances between them, compensate for time differences in the stimulation of sensory receptors. Hence the location of neurons, activated by the coincident arrival of multiple signals, reports the stimulus movement velocity. Despite its generality, most evidence for the place theory has been provided by studies of the auditory system of auditory specialists like the barn owl, but in the study of mammalian auditory systems the evidence is inconclusive. We ask to what extent the somatosensory systems of tactile specialists like rats and mice use distance dependent delays between neurons to compute the motion of tactile stimuli between the facial whiskers (or 'vibrissae'). We present a model in which synaptic inputs evoked by whisker deflections arrive at neurons in layer 2/3 (L2/3) somatosensory 'barrel' cortex at different times. The timing of synaptic inputs to each neuron depends on its location relative to sources of input in layer 4 (L4) that represent stimulation of each whisker. Constrained by the geometry and timing of projections from L4 to L2/3, the model can account for a range of experimentally measured responses to two-whisker stimuli. Consistent with that data, responses of model neurons located between the barrels to paired stimulation of two whiskers are greater than the sum of the responses to either whisker input alone. The model predicts that for neurons located closer to either barrel these supralinear responses are tuned for longer inter-whisker stimulation intervals, yielding a topographic map for the inter-whisker deflection interval across the surface of L2/3. This map constitutes a neural place code for the relative timing of sensory stimuli.
Project description:Mild to moderate traumatic brain injury (mTBI) leads to secondary neuronal loss via excitotoxic mechanisms, including mitochondrial Ca(2+) overload. However, in the surviving cellular population, mitochondrial Ca(2+) influx, and oxidative metabolism are diminished leading to suboptimal neuronal circuit activity and poor prognosis. Hence we tested the impact of boosting neuronal electrical activity and oxidative metabolism by facilitating mitochondrial Ca(2+) uptake in a rat model of mTBI. In developing rats (P25-P26) sustaining an mTBI, we demonstrate post-traumatic changes in cerebral blood flow (CBF) in the sensorimotor cortex in response to whisker stimulation compared to sham using functional Laser Doppler Imaging (fLDI) at adulthood (P67-P73). Compared to sham, whisker stimulation-evoked positive CBF responses decreased while negative CBF responses increased in the mTBI animals. The spatiotemporal CBF changes representing underlying neuronal activity suggested profound changes to neurovascular activity after mTBI. Behavioral assessment of the same cohort of animals prior to fLDI showed that mTBI resulted in persistent contralateral sensorimotor behavioral deficit along with ipsilateral neuronal loss compared to sham. Treating mTBI rats with Kaempferol, a dietary flavonol compound that enhanced mitochondrial Ca(2+) uptake, eliminated the inter-hemispheric asymmetry in the whisker stimulation-induced positive CBF responses and the ipsilateral negative CBF responses otherwise observed in the untreated and vehicle-treated mTBI animals in adulthood. Kaempferol also improved somatosensory behavioral measures compared to untreated and vehicle treated mTBI animals without augmenting post-injury neuronal loss. The results indicate that reduced mitochondrial Ca(2+) uptake in the surviving populations affect post-traumatic neural activation leading to persistent behavioral deficits. Improvement in sensorimotor behavior and spatiotemporal neurovascular activity following kaempferol treatment suggests that facilitation of mitochondrial Ca(2+) uptake in the early window after injury may sustain optimal neural activity and metabolism and contribute to improved function of the surviving cellular populations after mTBI.