ANTI-CD49D ANTIBODY TREATMENT IMPROVES SURVIVAL AND ATTENUATES NEUROCOGNITIVE DEFICITS AFTER TRAUMATIC BRAIN INJURY IN AGED MICE.

ABSTRACT: Patients 65 years and older account for an increasing proportion of traumatic brain injury (TBI) patients. Aged TBI patients experience increased morbidity and mortality compared with young TBI patients. We previously demonstrated a marked accumulation of CD8 + T-cells within the brains of aged TBI mice compared with young TBI mice. Therefore, we hypothesized that blocking peripheral T-cell infiltration into the injured brain would improve neurocognitive outcomes in aged mice after TBI. Young and aged male C57BL/6 mice underwent TBI via controlled cortical impact versus sham injury. Two hours after injuries, mice received an anti-CD49d antibody (aCD49d Ab) to block peripheral lymphocyte infiltration or its isotype control. Dosing was repeated every 2 weeks. Mortality was tracked. Neurocognitive testing for anxiety, associative learning, and memory was assessed. Motor function was evaluated. Plasma was collected for cytokine analysis. Flow cytometry was used to phenotype different immune cells within the brains. Consequently, aCD49d Ab treatment significantly improved post-TBI survival, anxiety level, associative learning, memory, and motor function in aged mice 2 months after TBI compared with isotype control treated mice. aCD49d Ab treatment augmented T H 2 response in the plasma of aged mice 2 months after TBI compared with isotype control-treated mice. Notably, aCD49d Ab treatment significantly reduced activated CD8 + cytotoxic T-cells within aged mouse brains after TBI. Contrastingly, no difference was detected in young mice after aCD49d Ab treatment. Collectively, aCD49 Ab treatment reduced T-cells in the injured brain, improved survival, and attenuated neurocognitive and gait deficits. Hence, aCD49d Ab may be a promising therapeutic intervention in aged TBI subjects-a population often excluded in TBI clinical trials.

The TgF344-AD rat: behavioral and proteomic changes associated with aging and protein expression in a transgenic rat model of Alzheimer's disease.

Animal models of Alzheimer's Disease (AD) are attractive tools for preclinical, prodromal drug testing. The TgF344-AD (Tg) rat exhibits cognitive deficits and 5 major hallmarks of AD. Here we show that spatial water maze (WMZ) memory deficits and proteomic differences in dorsal CA1 were present in young Tg rats. Aged learning-unimpaired (AU) and aged learning-impaired (AI) proteome associated changes were identified and differed by sex. Levels of phosphorylated tau, reactive astrocytes and microglia were significantly increased in aged Tg rats and correlated with the WMZ learning index (LI); in contrast, no significant correlation was present between amyloid plaques or insoluble Aß levels and LI. Neuroinflammatory markers were also significantly correlated with LI and increased in female Tg rats. The anti-inflammatory marker, triggering receptor expressed on myeloid cells-2 (TREM2), was significantly reduced in aged impaired Tg rats and correlated with LI. Identifying and understanding mechanisms that allow for healthy aging by overcoming genetic drivers for AD, and/or promoting drivers for successful aging, are important for developing successful therapeutics against AD.

Learning-related changes in cellular activity within mouse dentate gyrus during trace eyeblink conditioning.

Because the dentate gyrus serves as the first site for information processing in the hippocampal trisynaptic circuit, it an important structure for the formation of associative memories. Previous findings in rabbit had recorded populations of cells within dentate gyrus that may bridge the temporal gap between stimuli to support memory formation during trace eyeblink conditioning, an associative learning task. However, this previous work was unable to identify the types of cells demonstrating this type of activity. To explore these changes further, we did in vivo single-neuron recording in conjunction with physiological determination of cell types to investigate the functional role of granule cells, mossy cells, and interneurons in dentate gyrus during learning. Tetrode recordings were performed in young-adult mice during training on trace eyeblink conditioning, a hippocampal-dependent temporal associative memory task. Conditioned mice were able to successfully learn the task, with male mice learning at a faster rate than female mice. In the conditioned group, granule cells tended to show an increase in firing rate during conditioned stimulus presentation while mossy cells showed a decrease in firing rate during the trace interval and the unconditioned stimulus. Interestingly, populations of interneurons demonstrated learning-related increases and decreases in activity that began at onset of the conditioned stimulus and persisted through the trace interval. The current study also found a significant increase in theta power during stimuli presentation in conditioned animals, and this change in theta decreased over time. Ultimately, these data suggest unique involvement of granule cells, mossy cells, and interneurons in dentate gyrus in the formation of a trace associative memory. This work expands our knowledge of dentate gyrus function, helping to discern how aging and disease might disrupt this process.

Diet-induced Alzheimer's-like syndrome in the rabbit.

INTRODUCTION: Although mouse models of Alzheimer's disease (AD) have increased our understanding of the molecular basis of the disease, none of those models represent late-onset Alzheimer's Disease which accounts for >90% of AD cases, and no therapeutics developed in the mouse (with the possible exceptions of aduhelm/aducanumab and gantenerumab) have succeeded in preventing or reversing the disease. This technology has allowed much progress in understanding the molecular basis of AD. To further enhance our understanding, we used wild-type rabbit (with a nearly identical amino acid sequence for amyloid as in humans) to model LOAD by stressing risk factors including age, hypercholesterolemia, and elevated blood glucose levels (BGLs), upon an ε3-like isoform of apolipoprotein. We report a combined behavioral, imaging, and metabolic study using rabbit as a non-transgenic model to examine effects of AD-related risk factors on cognition, intrinsic functional connectivity, and magnetic resonance-based biomarkers of neuropathology. METHODS: Aging rabbits were fed a diet enriched with either 2% cholesterol or 10% fat/30% fructose. Monthly tests of novel object recognition (NOR) and object location memory (OLM) were administered to track cognitive impairment. Trace eyeblink conditioning (EBC) was administered as a final test of cognitive impairment. Magnetic resonance imaging (MRI) was used to obtain resting state connectivity and quantitative parametric data (R2*). RESULTS: Experimental diets induced hypercholesterolemia or elevated BGL. Both experimental diets induced statistically significant impairment of OLM (but not NOR) and altered intrinsic functional connectivity. EBC was more impaired by fat/fructose diet than by cholesterol. Whole brain and regional R2* MRI values were elevated in both experimental diet groups relative to rabbits on the control diet. DISCUSSION: We propose that mechanisms underlying LOAD can be assessed by stressing risk factors for inducing AD and that dietary manipulations can be used to assess etiological differences in the pathologies and effectiveness of potential therapeutics against LOAD. In addition, non-invasive MRI in awake, non-anesthetized rabbits further increases the translational value of this non-transgenic model to study AD.

Brain activity studied with magnetic resonance imaging in awake rabbits.

We reviewed fMRI experiments from our previous work in conscious rabbits, an experimental preparation that is advantageous for measuring brain activation that is free of anesthetic modulation and which can address questions in a variety of areas in sensory, cognitive, and pharmacological neuroscience research. Rabbits do not struggle or move for several hours while sitting with their heads restrained inside the horizontal bore of a magnet. This greatly reduces movement artifacts in magnetic resonance (MR) images in comparison to other experimental animals such as rodents, cats, and monkeys. We have been able to acquire high-resolution anatomic as well as functional images that are free of movement artifacts during several hours of restraint. Results from conscious rabbit fMRI studies with whisker stimulation are provided to illustrate the feasibility of this conscious animal model for functional MRI and the reproducibility of data gained with it.

Differential neuropathology and functional outcome after equivalent traumatic brain injury in aged versus young adult mice.

The CDC estimate that nearly 3 million Americans sustain a traumatic brain injury (TBI) each year. Even when medical comorbidities are accounted for, age is an independent risk factor for poor outcome after TBI. Nonetheless, few studies have examined the pathophysiology of age-linked biologic outcomes in TBI. We hypothesized that aged mice would demonstrate more severe neuropathology and greater functional deficits as compared to young adult mice after equivalent traumatic brain injuries. Young adult (14-week-old) and aged (80-week-old) C57BL/6 male mice underwent an open-head controlled cortical impact to induce TBI or a sham injury. At 30-days post-injury groups underwent behavioral phenotyping, magnetic resonance imaging, and histologic analyses. Contrary to our hypothesis, young adult TBI mice exhibited more severe neuropathology and greater loss of white matter connectivity as compared to aged mice after TBI. These findings correlated to differential functional outcomes in anxiety response, learning, and memory between young adult and aged mice after TBI. Although the mechanisms underlying this age-effect remain unclear, attenuated signs of secondary brain injury in aged TBI mice point towards different inflammatory and repair processes between age groups. These data suggest that age may need to be an a priori consideration in future clinical trial design.

Intact Female Mice Acquire Trace Eyeblink Conditioning Faster than Male and Ovariectomized Female Mice.

Female subjects have been widely excluded from past neuroscience work because of a number of biases, including the notion that cycling sex hormones increase variability. However, it is necessary to conduct behavioral research in mice that includes both sexes as mice are typically used for developing and evaluating future therapeutics. Understanding sex differences in learning is fundamental for the development of targeted therapies for numerous neurologic and neurodegenerative disorders, including Alzheimer's disease, which is more prevalent in females than males. This study set out to confirm the role of sex and necessity of circulating ovarian hormones in the acquisition of the temporal associative memory task trace eyeblink conditioning (tEBC) in C57BL/6J mice. We present evidence that sex and ovarian hormones are important factors in learning. Specifically, intact female mice learn significantly faster than both male and ovariectomized (ovx) female mice. Data from pseudoconditioned control mice indicate that sex differences are because of differences in learned associations, not sensitization or spontaneous blink rate. This study strengthens the idea that ovarian hormones such as estrogen and progesterone significantly influence learning and memory and that further research is needed to determine the underlying mechanisms behind their effects. Overall, our findings emphasize the necessity of including both sexes in future behavioral studies.

Cognitive aging is associated with redistribution of synaptic weights in the hippocampus.

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others' lifespans outlast their "mindspans."

Detection of memory- and learning-related brain connectivity changes following trace eyeblink-conditioning using resting-state functional magnetic resonance imaging in the awake rabbit.

Animal imaging studies have the potential to further establish resting-state fMRI (rs-fMRI) and enable its validation for clinical use. The rabbit subjects used in this work are an ideal model system for studying learning and behavior and are also an excellent established test subject for awake scanning given their natural tolerance for restraint. We found that analysis of rs-fMRI conducted on a cohort of rabbits undergoing eyeblink conditioning can reveal functional brain connectivity changes associated with learning, and that rs-fMRI can be used to capture differences between subjects with different levels of cognitive performance. rs-fMRI sessions were conducted on a cohort of rabbits before and after trace eyeblink conditioning. MRI results were analyzed using independent component analysis (ICA) and network analysis. Behavioral data were collected with standard methods using an infrared reflective sensor aimed at the cornea to detect blinks. Behavioral results were analyzed, and a median split was used to create two groups of rabbits based on their performance. The cohort of rabbits undergoing eyeblink conditioning exhibited increased functional connectivity in the cingulate cortex, retrosplenial cortex, and thalamus consistent with brain reorganization associated with increased learning. Differences in the striatum and right cerebellum were also identified between rabbits in the top or bottom halves of the group as measured by the behavioral assay. Thus, rs-fMRI can provide not only a tool to detect and monitor functional brain changes associated with learning, but also to discriminate between different levels of cognitive performance.

Aß oligomer induced cognitive impairment and evaluation of ACU193-MNS-based MRI in rabbit.

INTRODUCTION: Amyloid-beta oligomers (AßOs) accumulate in Alzheimer's disease and may instigate neuronal pathology and cognitive impairment. We examined the ability of a new probe for molecular magnetic resonance imaging (MRI) to detect AßOs in vivo, and we tested the behavioral impact of AßOs injected in rabbits, a species with an amino acid sequence that is nearly identical to the human sequence. METHODS: Intracerebroventricular (ICV) injection with stabilized AßOs was performed. Rabbits were probed for AßO accumulation using ACUMNS (an AßO-selective antibody [ACU193] coupled to magnetic nanostructures). Immunohistochemistry was used to verify AßO presence. Cognitive impairment was evaluated using object location and object recognition memory tests and trace eyeblink conditioning. RESULTS: AßOs in the entorhinal cortex of ICV-injected animals were detected by MRI and confirmed by immunohistochemistry. Injections of AßOs also impaired hippocampal-dependent, but not hippocampal-independent, tasks and the area fraction of bound ACUMNs correlated with the behavioral impairment. DISCUSSION: Accumulation of AßOs can be visualized in vivo by MRI of ACUMNS and the cognitive impairment induced by the AßOs can be followed longitudinally with the novel location memory test.

Intrinsic Excitability Increase in Cerebellar Purkinje Cells after Delay Eye-Blink Conditioning in Mice.

Cerebellar-based learning is thought to rely on synaptic plasticity, particularly at synaptic inputs to Purkinje cells. Recently, however, other complementary mechanisms have been identified. Intrinsic plasticity is one such mechanism, and depends in part on the downregulation of calcium-dependent SK-type K+ channels, which contribute to a medium-slow afterhyperpolarization (AHP) after spike bursts, regulating membrane excitability. In the hippocampus, intrinsic plasticity plays a role in trace eye-blink conditioning; however, corresponding excitability changes in the cerebellum in associative learning, such as in trace or delay eye-blink conditioning, are less well studied. Whole-cell patch-clamp recordings were obtained from Purkinje cells in cerebellar slices prepared from male mice ∼48 h after they learned a delay eye-blink conditioning task. Over a period of repeated training sessions, mice received either paired trials of a tone coterminating with a periorbital shock (conditioning) or trials in which these stimuli were randomly presented in an unpaired manner (pseudoconditioning). Purkinje cells from conditioned mice show a significantly reduced AHP after trains of parallel fiber stimuli and after climbing fiber evoked complex spikes. The number of spikelets in the complex spike waveform is increased after conditioning. Moreover, we find that SK-dependent intrinsic plasticity is occluded in conditioned, but not pseudoconditioned mice. These findings show that excitability is enhanced in Purkinje cells after delay eye-blink conditioning, and point toward a downregulation of SK channels as a potential underlying mechanism. The observation that this learning effect lasts at least up to 2 d after training shows that intrinsic plasticity regulates excitability in the long term.SIGNIFICANCE STATEMENT Plasticity of membrane excitability ("intrinsic plasticity") has been observed in invertebrate and vertebrate neurons, coinduced with synaptic plasticity or in isolation. Although the cellular phenomenon per se is well established, it remains unclear what role intrinsic plasticity plays in learning and if it even persists long enough to serve functions in engram physiology beyond aiding synaptic plasticity. Here, we demonstrate that cerebellar Purkinje cells upregulate excitability in delay eye-blink conditioning, a form of motor learning. This plasticity is observed 48 h after training and alters synaptically evoked spike firing and integrative properties of these neurons. These findings show that intrinsic plasticity enhances the spike firing output of Purkinje cells and persists over the course of days.

Conditioned Contextual Freezing is A Neurobehavioral Biomarker of Axonal Injury Indicated by Reduced Fractional Anisotropy in A Mouse Model of Blast-Induced Mild Traumatic Brain Injury.

Mild traumatic brain injury (TBI) is an important public health problem generated by closed head injury. This study is focused on the impact of blast-induced mild TBI on auditory trace and delay fear conditioning, models of declarative and non-declarative memory, respectively, and the correlation of conditioned freezing and fractional anisotropy, a measure of axonal state. A supersonic helium pressure wave was generated by a shock tube to blast 8-week-old male mice on Day 1 for 1.4 msec with an incident pressure of 16 psi, corresponding to a reflected pressure of 56.9 psi at the mouse head. On Day 3, the mice were subjected to auditory trace- or delay-fear conditioning. On Day 4, contextual freezing in the trained context, and precue and cued freezing in a novel context were determined. After cardiac perfusion on Day 5, ex vivo images were obtained with diffusion tensor imaging at 14.1 Tesla. We observed that delay fear conditioning prevented or reversed the decrease in fractional anisotropy in both the medial and lateral corpus callosum suggesting axonal stabilization of potentially behavioral therapeutic significance. Moderately strong and statistically significant Pearson correlations were found between fractional anisotropy and contextual freezing in the medial and lateral corpus callosum of blasted and sham-blasted delay- or trace-fear conditioned mice. Thus, contextual freezing is a neurobehavioral biomarker for axonal injury in mild TBI and is a reliable and high-throughput behavioral assay for the evaluation of potential therapeutics to treat mild TBI.

Genetic Ablation of Neural Progenitor Cells Impairs Acquisition of Trace Eyeblink Conditioning.

Adult-born neurons are believed to play a role in memory formation by providing enhanced plasticity to the hippocampus. Past studies have demonstrated that reduction of neurogenesis impairs associative learning, but these experiments used irradiation or neurotoxic substances, which may have had unintended off-target effects. Therefore, to investigate the role of these adult-born neurons more precisely, we used nestin-HSV-TK transgenic mice (Nes-TK) to selectively ablate newborn neurons. Nes-TK mice were fed a chow infused with valganciclovir to induce the ablation of neural progenitor cells. After being on this diet for 4 weeks, mice were trained on trace eyeblink conditioning, a hippocampus-dependent temporal associative memory task. Following the completion of training, brain sections from these animals were stained for doublecortin, a marker for immature neurons, to quantify levels of neurogenesis. We found that male transgenic mice on valganciclovir had significantly decreased amounts of doublecortin relative to male control animals, indicating a successful reduction in levels of neurogenesis. In conjunction with this reduction in neurogenesis, the male transgenic mice on valganciclovir learned at a significantly slower rate than male control mice. The female Nes-TK mice on valganciclovir showed no significant decrease in neurogenesis and no behavioral impairment relative to female control mice. Ultimately, the results are consistent with, and expand upon, prior studies that demonstrated that adult-born neurons are involved in the formation of associative memories. This study also provides a foundation to continue to explore the physiological role of newborn neurons with in vivo recordings during behavioral training.

Differential responsivity of neurons in perirhinal cortex, lateral entorhinal cortex, and dentate gyrus during time-bridging learning.

Many studies have focused on the function of hippocampal region CA1 as a critical site for associative memory, but much less is known about changes in the afferents to CA1. Here we report the activity of multiple single neurons from perirhinal and entorhinal cortex and from dentate gyrus during trace eyeblink conditioning as well as consolidated recall, and in pseudo-conditioned control rabbits. We also report an analysis of theta activity filtered from the local field potential (LFP). Our results show early associative changes in single-neuron firing rate as well as theta oscillations in lateral entorhinal cortex (EC) and dentate gyrus (DG), and increases in the number of responsive neurons in perirhinal cortex. In both EC and DG, a subset of neurons from conditioned animals exhibited an elevated baseline firing rate and large responses to the conditioned stimulus and trace period. A similar population of cells has been seen in DG and in medial, but not lateral, EC during spatial tasks, suggesting that lateral EC contains cells responsive to a temporal associative task. In contrast to recent studies in our laboratory that found significant CA1 contributions to long-term memory, the activity profiles of neurons within EC and DG were similar for conditioned and pseudoconditioned rabbits during post-consolidation sessions. Collectively these results demonstrate that individual subregions of medial temporal lobe differentially support new and remotely acquired memories. Neuron firing profiles were similar on training trials when conditioned responses were and were not exhibited, demonstrating that these temporal lobe regions represent the CS-US association and do not control the behavioral response. The analysis of theta activity revealed that theta power was modulated by the conditioning stimuli in both the conditioned and pseudoconditioned groups and that although both groups exhibited a resetting of phase to the corneal airpuff, only the conditioned group exhibited a resetting of phase to the whisker conditioned stimulus.

The rabbit as a behavioral model system for magnetic resonance imaging.

BACKGROUND: fMRI requires that subjects not move during image acquisition. This has been achieved by instructing people not to move, or by anesthetizing experimental animal subjects to induce immobility. We have demonstrated that a surgically implanted headbolt onto the skull of a rabbit allows their brain to be imaged comfortably while the animal is awake. This article provides a detailed method for the preparation. NEW METHOD: We took advantage of the rabbit's tolerance for restraint to image the brain while holding the head at the standard stereotaxic angle. Visual stimulation was produced by flashing green LEDs and whisker stimulation was done by powering a small coil of wire attached to a fiber band. Blinking was recorded with an infrared emitter/detector directed at the eye with fiber-optic cabling. RESULTS: Results indicate that a single daily session of habituation is sufficient to produce adequate immobility on subsequent days to avoid movement artifacts. Results include high resolution images in the stereotaxic plane of the rabbit. COMPARISON WITH EXISTING METHOD(S): We see no degradation or distortion of MR signal, and the headbolt provides a means for rapid realignment of the head in the magnet from day to day, and across subjects. The use of rabbits instead of rodents allows much shorter periods of habituation, and the rabbit allows behavior to be observed during the day while the animal is in its normal wake cycle. CONCLUSIONS: The natural tolerance of the rabbit for restraint makes it a valuable subject for MRI studies of the brain.

Non-comedogenic and non-acnegenic claim substantiation.

There are currently two methods to evaluate comedogenecity. One is the inexpensive human model developed by Mills and Kligman and modified by others. The second is the more costly human clinical trial, which is the gold standard for comedogenesis and to which the human model is compared. The qualification of each method to support the comedogenecity claim is evaluated and contrasted.

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

Pretrial functional connectivity differentiates behavioral outcomes during trace eyeblink conditioning in the rabbit.

Fluctuations in neural activity can produce states that facilitate and accelerate task-related performance. Acquisition of trace eyeblink conditioning (tEBC) in the rabbit is enhanced when trials are contingent on optimal pretrial activity in the hippocampus. Other regions which are essential for whisker-signaled tEBC, such as the cerebellar interpositus nucleus (IPN), somatosensory and prelimbic cortices, may also show optimal connectivity prior to successful performance. Functional magnetic resonance imaging (fMRI) was acquired in nine rabbits during tEBC on the first and tenth days of initial training and once again after a 30-d, training-free hiatus. Data acquired during the intertrial interval was parsed depending on whether or not a conditioned response (CR) occurred on the upcoming trial and seed-based functional connectivity was calculated among the IPN, hippocampus, somatosensory, and prelimbic cortices. Functional connectivity between the left somatosensory cortex and right IPN, regions critical for establishing and producing CRs evoked by right vibrissae vibration and right corneal airpuff, was significantly negative prior to successful, CR trials as compared with unsuccessful, non-CR trials. Differences were not observed for any of the other possible combinations of connectivity. Our results demonstrate that specific pretrial functional connectivity exists within the rabbit brain and differentiates between upcoming behavioral response outcomes. Online analysis of network fluctuations has the potential to be used as the basis for therapeutic interventions to facilitate learning and memory.

Intrinsic connectivity of neural networks in the awake rabbit.

The way in which the brain is functionally connected into different networks has emerged as an important research topic in order to understand normal neural processing and signaling. Since some experimental manipulations are difficult or unethical to perform in humans, animal models are better suited to investigate this topic. Rabbits are a species that can undergo MRI scanning in an awake and conscious state with minimal preparation and habituation. In this study, we characterized the intrinsic functional networks of the resting New Zealand White rabbit brain using BOLD fMRI data. Group independent component analysis revealed seven networks similar to those previously found in humans, non-human primates and/or rodents including the hippocampus, default mode, cerebellum, thalamus, and visual, somatosensory, and parietal cortices. For the first time, the intrinsic functional networks of the resting rabbit brain have been elucidated demonstrating the rabbit's applicability as a translational animal model. Without the confounding effects of anesthetics or sedatives, future experiments may employ rabbits to understand changes in neural connectivity and brain functioning as a result of experimental manipulation (e.g., temporary or permanent network disruption, learning-related changes, and drug administration).

Activity-induced manganese-dependent MRI (AIM-MRI) and functional MRI in awake rabbits during somatosensory stimulation.

Activity-induced manganese-dependent MRI (AIM-MRI) is a powerful tool to track system-wide neural activity using high resolution, quantitative T1-weighted MRI in animal models and has significant advantages for investigating neural activity over other modalities including BOLD fMRI. With AIM-MRI, Mn(2+) ions enter neurons via voltage-gated calcium channels preferentially active during the time of experimental exposure. A broad range of AIM-MRI studies using different species studying different phenomena have been performed, but few of these studies provide a systematic evaluation of the factors influencing the detection of Mn(2+) such as dosage and the temporal characteristics of Mn(2+) uptake. We identified an optimal dose of Mn(2+) (25 mg/kg, s.c.) in order to characterize the time-course of Mn(2+) accumulation in active neural regions in the rabbit. T1-weighted MRI and functional MRI were collected 0-3, 6-9, and 24-27 h post-Mn(2+) injection while the vibrissae on the right side were vibrated. Significant BOLD activation in the left somatosensory (SS) cortex and left ventral posteromedial (VPM) thalamic nucleus was detected during whisker vibration. T1-weighted signal intensities were extracted from these regions, their corresponding contralateral regions and the visual cortex (to serve as controls). A significant elevation in T1-weighted signal intensity in the left SS cortex (relative to right) was evident 6-9 and 24-27 h post-Mn(2+) injection while the left VPM thalamus showed a significant enhancement (relative to the right) only during the 24-27 h session. Visual cortex showed no hemispheric difference at any timepoint. Our results suggest that studies employing AIM-MRI would benefit by conducting experimental manipulations 6-24 h after subcutaneous MnCl2 injections to optimize the concentration of contrast agent in the regions active during the exposure.