Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Anterior cingulate cortex-related functional hyperconnectivity underlies sensory hypersensitivity in Grin2b-mutant mice.

Sensory abnormalities are observed in ~90% of individuals with autism spectrum disorders (ASD), but the underlying mechanisms are poorly understood. GluN2B, an NMDA receptor subunit that regulates long-term depression and circuit refinement during brain development, has been strongly implicated in ASD, but whether GRIN2B mutations lead to sensory abnormalities remains unclear. Here, we report that Grin2b-mutant mice show behavioral sensory hypersensitivity and brain hyperconnectivity associated with the anterior cingulate cortex (ACC). Grin2b-mutant mice with a patient-derived C456Y mutation (Grin2bC456Y/+) show sensory hypersensitivity to mechanical, thermal, and electrical stimuli through supraspinal mechanisms. c-fos and functional magnetic resonance imaging indicate that the ACC is hyperactive and hyperconnected with other brain regions under baseline and stimulation conditions. ACC pyramidal neurons show increased excitatory synaptic transmission. Chemogenetic inhibition of ACC pyramidal neurons normalizes ACC hyperconnectivity and sensory hypersensitivity. These results suggest that GluN2B critically regulates ASD-related cortical connectivity and sensory brain functions.

Dissection of brain-wide resting-state and functional somatosensory circuits by fMRI with optogenetic silencing.

To further advance functional MRI (fMRI)-based brain science, it is critical to dissect fMRI activity at the circuit level. To achieve this goal, we combined brain-wide fMRI with neuronal silencing in well-defined regions. Since focal inactivation suppresses excitatory output to downstream pathways, intact input and suppressed output circuits can be separated. Highly specific cerebral blood volume-weighted fMRI was performed with optogenetic stimulation of local GABAergic neurons in mouse somatosensory regions. Brain-wide spontaneous somatosensory networks were found mostly in ipsilateral cortical and subcortical areas, which differed from the bilateral homotopic connections commonly observed in resting-state fMRI data. The evoked fMRI responses to somatosensory stimulation in regions of the somatosensory network were successfully dissected, allowing the relative contributions of spinothalamic (ST), thalamocortical (TC), corticothalamic (CT), corticocortical (CC) inputs, and local intracortical circuits to be determined. The ventral posterior thalamic nucleus receives ST inputs, while the posterior medial thalamic nucleus receives CT inputs from the primary somatosensory cortex (S1) with TC inputs. The secondary somatosensory cortex (S2) receives mostly direct CC inputs from S1 and a few TC inputs from the ventral posterolateral nucleus. The TC and CC input layers in cortical regions were identified by laminar-specific fMRI responses with a full width at half maximum of <150 µm. Long-range synaptic inputs in cortical areas were amplified approximately twofold by local intracortical circuits, which is consistent with electrophysiological recordings. Overall, whole-brain fMRI with optogenetic inactivation revealed brain-wide, population-based, long-range circuits, which could complement data typically collected in conventional microscopic functional circuit studies.

Early fMRI responses to somatosensory and optogenetic stimulation reflect neural information flow.

Blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has been widely used to localize brain functions. To further advance understanding of brain functions, it is critical to understand the direction of information flow, such as thalamocortical versus corticothalamic projections. For this work, we performed ultrahigh spatiotemporal resolution fMRI at 15.2 T of the mouse somatosensory network during forepaw somatosensory stimulation and optogenetic stimulation of the primary motor cortex (M1). Somatosensory stimulation induced the earliest BOLD response in the ventral posterolateral nucleus (VPL), followed by the primary somatosensory cortex (S1) and then M1 and posterior thalamic nucleus. Optogenetic stimulation of excitatory neurons in M1 induced the earliest BOLD response in M1, followed by S1 and then VPL. Within S1, the middle cortical layers responded to somatosensory stimulation earlier than the upper or lower layers, whereas the upper cortical layers responded earlier than the other two layers to optogenetic stimulation in M1. The order of early BOLD responses was consistent with the canonical understanding of somatosensory network connections and cannot be explained by regional variabilities in the hemodynamic response functions measured using hypercapnic stimulation. Our data demonstrate that early BOLD responses reflect the information flow in the mouse somatosensory network, suggesting that high-field fMRI can be used for systems-level network analyses.

Miniaturized MR-compatible ultrasound system for real-time monitoring of acoustic effects in mice using high-resolution MRI.

Visualization of focused ultrasound in high spatial and temporal resolution is crucial for accurately and precisely targeting brain regions noninvasively. Magnetic resonance imaging (MRI) is the most widely used noninvasive tool for whole-brain imaging. However, focused ultrasound studies employing high-resolution (> 9.4 T) MRI in small animals are limited by the small size of the radiofrequency (RF) volume coil and the noise sensitivity of the image to external systems such as bulky ultrasound transducers. This technical note reports a miniaturized ultrasound transducer system packaged directly above a mouse brain for monitoring ultrasound-induced effects using high-resolution 9.4 T MRI. Our miniaturized system integrates MR-compatible materials with electromagnetic (EM) noise reduction techniques to demonstrate echo-planar imaging (EPI) signal changes in the mouse brain at various ultrasound acoustic intensities. The proposed ultrasound-MRI system will enable extensive research in the expanding field of ultrasound therapeutics.

Theory of chemical exchange saturation transfer MRI in the context of different magnetic fields.

The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 11, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:11 on Wiley Online Library: https://doi.org/10.1002/nbm.4789.

Autodisplay of streptococcal protein G for construction of an orientation-controlled immunoaffinity layer.

An immunoaffinity layer with orientation-controlled antibodies was constructed to express streptococcal protein G in Escherichia coli cells using autodisplay technology. The sequence of protein G, a specific IgG-binding protein, was inserted into the autodisplay vector using recombinant technology and the constructed plasmid vector was transformed into E. coli cells. Protein G was confirmed to be autodisplayed with a high density of 2 × 105 copies per cell by SDS-PAGE analysis, and its IgG-binding affinity was confirmed by fluorescence microscopy. Autodisplayed protein G showed higher affinity than the IgG-binding Z-domain for goat IgG. Immunoassays based on E. coli cells were established to detect horseradish peroxidase (HRP) and C-reactive protein (CRP). Protein G autodisplaying E. coli cells were utilized as a solid support and immunoassays showed improved sensitivity by orientation control of autodisplayed protein G. The outer membrane (OM) of protein G autodisplaying E. coli was isolated and layered to construct an immunoaffinity layer. The OM was coated on a microplate to perform the immunoassays, which showed limits of detection of 5 and 0.2 ng mL-1 for HRP and CRP, respectively. An OM layer with autodisplayed protein G was applied as the immunoaffinity layer of a surface plasmon resonance (SPR) biosensor. After CRP detection, the SPR responses showed good linearity, with an R2 value of 0.99. The immunoaffinity layer with orientation control by autodisplayed protein G was confirmed to be applicable in immunoassays and immunosensors to improve sensitivity.

Deep brain stimulation of the anterior nuclei of the thalamus can alleviate seizure severity and induce hippocampal GABAergic neuronal changes in a pilocarpine-induced epileptic mouse brain.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) has been widely used as an effective treatment for refractory temporal lobe epilepsy. Despite its promising clinical outcome, the exact mechanism of how ANT-DBS alleviates seizure severity has not been fully understood, especially at the cellular level. To assess effects of DBS, the present study examined electroencephalography (EEG) signals and locomotor behavior changes and conducted immunohistochemical analyses to examine changes in neuronal activity, number of neurons, and neurogenesis of inhibitory neurons in different hippocampal subregions. ANT-DBS alleviated seizure activity, abnormal locomotor behaviors, reduced theta-band, increased gamma-band EEG power in the interictal state, and increased the number of neurons in the dentate gyrus (DG). The number of parvalbumin- and somatostatin-expressing inhibitory neurons was recovered to the level in DG and CA1 of naïve mice. Notably, BrdU-positive inhibitory neurons were increased. In conclusion, ANT-DBS not only could reduce the number of seizures, but also could induce neuronal changes in the hippocampus, which is a key region involved in chronic epileptogenesis. Importantly, our results suggest that ANT-DBS may lead to hippocampal subregion-specific cellular recovery of GABAergic inhibitory neurons.

Improved laminar specificity and sensitivity by combining SE and GE BOLD signals.

The most widely used gradient-echo (GE) blood oxygenation level-dependent (BOLD) contrast has high sensitivity, but low specificity due to draining vein contributions, while spin-echo (SE) BOLD approach at ultra-high magnetic fields is highly specific to neural active sites but has lower sensitivity. To obtain high specificity and sensitivity, we propose to utilize a vessel-size-sensitive filter to the GE-BOLD signal, which suppresses macrovascular contributions and to combine selectively retained microvascular GE-BOLD signals with the SE-BOLD signals. To investigate our proposed idea, fMRI with 0.8 mm isotropic resolution was performed on the primary motor and sensory cortices in humans at 7 T by implementing spin- and gradient-echo (SAGE) echo planar imaging (EPI) acquisition. Microvascular-passed sigmoidal filters were designed based upon the vessel-size-sensitive ΔR2*/ΔR2 value for retaining GE-BOLD signals originating from venous vessels with ≤ 45 µm and ≤ 65 µm diameter. Unlike GE-BOLD fMRI, the laminar profile of SAGE-BOLD fMRI with the vessel-size-sensitive filter peaked at ∼ 1.0 mm from the surface of the primary motor and sensory cortices, demonstrating an improvement of laminar specificity over GE-BOLD fMRI. Also, the functional sensitivity of SAGE BOLD at middle layers (0.75-1.5 mm) was improved by ∼ 80% to ∼100% when compared with SE BOLD. In summary, we showed that combined GE- and SE-BOLD fMRI with the vessel-size-sensitive filter indeed yielded improved laminar specificity and sensitivity and is therefore an excellent tool for high spatial resolution ultra-high filed (UHF)-fMRI studies for resolving mesoscopic functional units.

Role of chemical exchange on the relayed nuclear Overhauser enhancement signal in saturation transfer MRI.

PURPOSE: The pH sensitivity of chemical exchange-relayed nuclear Overhauser enhancement (rNOE) signal in a saturation transfer experiment is not fully understood and needs further investigation. METHODS: A three-pool-exchange model was simulated assuming that the magnetization transfer between an NOE pool and water is relayed by a chemical exchange (CE) pool. The saturation transfer signals from bovine serum albumin (BSA) and egg white albumin (EWA) phantoms were measured with different pH or different D2 O/H2 O mixture solutions. RESULTS: Simulation results showed that the rNOE signal is independent of the Larmor frequency of the CE pool, indicating any CE pool can effectively relay NOE magnetization. The rNOE signal is sensitive to a change of the CE pool size and/or exchange rate only if the CE becomes a rate-limiting step in the relay process. The rNOE signal from BSA phantoms showed larger pH-dependence at -3.0 ppm than those at -1.9 and -4.0 ppm. However, rNOE signals from aliphatic protons have much weaker pH-dependence than the CEST signal, suggesting that CE is unlikely the rate-limiting step and the rNOE signals in BSA are mainly relayed by fast exchanging protons. The existence of aromatic NOE was confirmed by proton spectroscopy. CONCLUSION: The pH-sensitivity of the rNOE signal is determined by whether the CE process is a rate-limiting step in the relay. The rNOE signal has much weaker pH-sensitivity than the CEST signal in BSA proteins, which can explain the weak pH sensitivity of rNOE in vivo.

Review and consensus recommendations on clinical APT-weighted imaging approaches at 3T: Application to brain tumors.

Amide proton transfer-weighted (APTw) MR imaging shows promise as a biomarker of brain tumor status. Currently used APTw MRI pulse sequences and protocols vary substantially among different institutes, and there are no agreed-on standards in the imaging community. Therefore, the results acquired from different research centers are difficult to compare, which hampers uniform clinical application and interpretation. This paper reviews current clinical APTw imaging approaches and provides a rationale for optimized APTw brain tumor imaging at 3 T, including specific recommendations for pulse sequences, acquisition protocols, and data processing methods. We expect that these consensus recommendations will become the first broadly accepted guidelines for APTw imaging of brain tumors on 3 T MRI systems from different vendors. This will allow more medical centers to use the same or comparable APTw MRI techniques for the detection, characterization, and monitoring of brain tumors, enabling multi-center trials in larger patient cohorts and, ultimately, routine clinical use.

Theory of chemical exchange saturation transfer MRI in the context of different magnetic fields.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a versatile MRI method that provides contrast based on the level of molecular and metabolic activity. This contrast arises from indirect measurement of protons in low concentration molecules that are exchanging with the abundant water proton pool. The indirect measurement is based on magnetization transfer of radio frequency (rf)-prepared magnetization from the small pool to the water pool. The signal can be modeled by the Bloch-McConnell equations combining standard magnetization dynamics and chemical exchange processes. In this article, we review analytical solutions of the Bloch-McConnell equations and especially the derived CEST signal equations and their implications. The analytical solutions give direct insight into the dependency of measurable CEST effects on underlying parameters such as the exchange rate and concentration of the solute pools, but also on the system parameters such as the rf irradiation field B1 , as well as the static magnetic field B0 . These theoretical field-strength dependencies and their influence on sequence design are highlighted herein. In vivo results of different groups making use of these field-strength benefits/dependencies are reviewed and discussed.

Contribution of Excitatory and Inhibitory Neuronal Activity to BOLD fMRI.

The BOLD fMRI response in the cortex is often assumed to reflect changes in excitatory neural activity. However, the contribution of inhibitory neurons to BOLD fMRI is unclear. Here, the role of inhibitory and excitatory activity was examined using multimodal approaches: electrophysiological recording, 15.2 T fMRI, optical intrinsic signal imaging, and modeling. Inhibitory and excitatory neuronal activity in the somatosensory cortex were selectively modulated by 20-s optogenetic stimulation of VGAT-ChR2 and CaMKII-ChR2 mice, respectively. Somatosensory stimulation and optogenetic stimulation of excitatory neurons induced positive BOLD responses in the somatosensory network, whereas stimulation of inhibitory neurons produced biphasic responses at the stimulation site, initial positive and later negative BOLD signals, and negative BOLD responses at downstream sites. When the stimulation duration was reduced to 5 s, the hemodynamic response of VGAT-ChR2 mice to optogenetic stimulation was only positive. Lastly, modeling performed from neuronal and hemodynamic data shows that the hemodynamic response function (HRF) of excitatory neurons is similar across different conditions, whereas the HRF of inhibitory neurons is highly sensitive to stimulation frequency and peaks earlier than that of excitatory neurons. Our study provides insights into the neurovascular coupling of excitatory and inhibitory neurons and the interpretation of BOLD fMRI signals.

Optogenetic fMRI for Brain-Wide Circuit Analysis of Sensory Processing.

Sensory processing is a complex neurological process that receives, integrates, and responds to information from one's own body and environment, which is closely related to survival as well as neurological disorders. Brain-wide networks of sensory processing are difficult to investigate due to their dynamic regulation by multiple brain circuits. Optogenetics, a neuromodulation technique that uses light-sensitive proteins, can be combined with functional magnetic resonance imaging (ofMRI) to measure whole-brain activity. Since ofMRI has increasingly been used for investigating brain circuits underlying sensory processing for over a decade, we systematically reviewed recent ofMRI studies of sensory circuits and discussed the challenges of optogenetic fMRI in rodents.

Thrombin Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage Induced Neonatal Rats.

Severe intraventricular hemorrhage (IVH) remains a major cause of high mortality and morbidity in extremely preterm infants. Mesenchymal stem cell (MSC) transplantation is a possible therapeutic option, and development of therapeutics with enhanced efficacy is necessary. This study investigated whether thrombin preconditioning improves the therapeutic efficacy of human Wharton's jelly-derived MSC transplantation for severe neonatal IVH, using a rat model. Severe neonatal IVH was induced by injecting 150 µL blood into each lateral ventricle on postnatal day (P) 4 in Sprague-Dawley rats. After 2 days (P6), naïve MSCs or thrombin-preconditioned MSCs (1 × 105/10 µL) were transplanted intraventricularly. After behavioral tests, brain tissues and cerebrospinal fluid of P35 rats were obtained for histological and biochemical analyses, respectively. Thrombin-preconditioned MSC transplantation significantly reduced IVH-induced ventricular dilatation on in vivo magnetic resonance imaging, which was coincident with attenuations of reactive gliosis, cell death, and the number of activated microglia and levels of inflammatory cytokines after IVH induction, compared to naïve MSC transplantation. In the behavioral tests, the sensorimotor and memory functions significantly improved after transplantation of thrombin-preconditioned MSCs, compared to naïve MSCs. Overall, thrombin preconditioning significantly improves the therapeutic potential and more effectively attenuates brain injury, including progressive ventricular dilatation, gliosis, cell death, inflammation, and neurobehavioral functional impairment, in newborn rats with induced severe IVH than does naïve MSC transplantation.

Excitation-Inhibition Imbalance Leads to Alteration of Neuronal Coherence and Neurovascular Coupling under Acute Stress.

A single stressful event can cause morphologic and functional changes in neurons and even malfunction of vascular systems, which can lead to acute stress disorder or post-traumatic stress disorder. However, there is a lack of evidence regarding how acute stress impacts neuronal activity, the concurrent vascular response, and the relationship between these two factors, which is defined as neurovascular coupling. Here, using in vivo two-photon imaging, we found that NMDA-evoked calcium transients of excitatory neurons were impaired and that vasodilation of penetrating arterioles was concomitantly disrupted in acutely stressed male mice. Furthermore, acute stress altered the relationship between excitatory neuronal calcium coherence and vascular responses. By measuring NMDA-evoked excitatory and inhibitory neuronal calcium activity in acute brain slices, we confirmed that neuronal coherence both between excitatory neurons and between excitatory and inhibitory neurons was reduced by acute stress but restored by blockade of glucocorticoid receptor signaling. Furthermore, the ratio of sEPSCs to sIPSCs was altered by acute stress, suggesting that the excitation-inhibition balance was disrupted by acute stress. In summary, in vivo, ex vivo, and whole-cell recording studies demonstrate that acute stress modifies excitatory-inhibitory neuronal coherence, disrupts the excitation-inhibition balance, and causes consequent neurovascular coupling changes, providing critical insights into the neural mechanism of stress-induced disorders.SIGNIFICANCE STATEMENT Acute stress can cause pathologic conditions, such as acute stress disorder and post-traumatic stress disorder, by affecting the functions of neurons and blood vessels. However, investigations into the impacts of acute stress on neurovascular coupling, the tight connection between local neural activity and subsequent blood flow changes, are lacking. Through investigations at the in vivo, ex vivo, and whole-cell recording levels, we found that acute stress alters the NMDA-evoked vascular response, impairs the function and coherence of excitatory and inhibitory neurons, and disrupts the excitatory and inhibitory balance. These novel findings provide insights into the relevance of the excitatory-inhibitory balance, neuronal coherence, and neurovascular coupling to stress-induced disorders.

Improvement of sensitivity and specificity for laminar BOLD fMRI with double spin-echo EPI in humans at 7 T.

Mapping mesoscopic cortical functional units such as columns or laminae is increasingly pursued by ultra-high field (UHF) functional magnetic resonance imaging (fMRI). The most popular approach for high-resolution fMRI is currently gradient-echo (GE) blood oxygenation level-dependent (BOLD) fMRI. However, its spatial accuracy is reduced due to its sensitivity to draining vessels, including pial veins, whereas spin-echo (SE) BOLD signal is expected to have higher spatial accuracy, albeit with lower sensitivity than the GE-BOLD signal. Here, we introduce a new double spin-echo (dSE) echo-planar imaging (EPI) method to improve the sensitivity of SE-BOLD contrast by averaging two spin-echoes using three radiofrequency pulses. Human fMRI experiments were performed with slices perpendicular to the central sulcus between motor and sensory cortices at 7 T during fist-clenching with touching. First, we evaluated the feasibility of single-shot dSE-EPI for BOLD fMRI with 1.5 mm isotropic resolution and found that dSE-BOLD fMRI has higher signal-to-noise ratio (SNR), temporal SNR (tSNR), and higher functional sensitivity than conventional SE-BOLD fMRI. Second, to investigate the laminar specificity of dSE-BOLD fMRI, we implemented a multi-shot approach to achieve 0.8-mm isotropic resolution with sliding-window reconstruction. Unlike GE-BOLD fMRI, the cortical profile of dSE-BOLD fMRI peaked at ~ 1.0 mm from the surface of the primary motor and sensory cortices, demonstrating an improvement of laminar specificity in humans over GE-BOLD fMRI. The proposed multi-shot dSE-EPI method is viable for high spatial resolution UHF-fMRI studies in the pursuit of resolving mesoscopic functional units.

Postsynaptic activity of inhibitory neurons evokes hemodynamic fMRI responses.

Functional MRI responses are localized to the synaptic sites of evoked inhibitory neurons, but it is unknown whether, or by what mechanisms, these neurons initiate functional hyperemia. Here, the neuronal origins of these hemodynamic responses were investigated by fMRI or local field potential and blood flow measurements during topical application of pharmacological agents when GABAergic granule cells in the rat olfactory bulb were synaptically targeted. First, to examine if postsynaptic activation of these inhibitory neurons was required for neurovascular coupling, we applied an NMDA receptor antagonist during cerebral blood volume-weighted fMRI acquisition and found that responses below the drug application site (up to ~1.5 mm) significantly decreased within ~30 min. Similarly, large decreases in granule cell postsynaptic activities and blood flow responses were observed when AMPA or NMDA receptor antagonists were applied. Second, inhibition of nitric oxide synthase preferentially decreased the initial, fast component of the blood flow response, while inhibitors of astrocyte-specific glutamate transporters and vasoactive intestinal peptide receptors did not decrease blood flow responses. Third, inhibition of GABA release with a presynaptic GABAB receptor agonist caused less reduction of neuronal and blood flow responses compared to the postsynaptic glutamate receptor antagonists. In conclusion, local hyperemia by synaptically-evoked inhibitory neurons was primarily driven by their postsynaptic activities, possibly through NMDA receptor-dependent calcium signaling that was not wholly dependent on nitric oxide.

Characteristics of fMRI responses to visual stimulation in anesthetized vs. awake mice.

The functional characteristics of the mouse visual system have not previously been well explored using fMRI. In this research, we examined 9.4 T BOLD fMRI responses to visual stimuli of varying pulse durations (1 - 50 ms) and temporal frequencies (1 - 10 Hz) under ketamine and xylazine anesthesia, and compared fMRI responses of anesthetized and awake mice. Under anesthesia, significant positive BOLD responses were detected bilaterally in the major structures of the visual pathways, including the dorsal lateral geniculate nuclei, superior colliculus, lateral posterior nucleus of thalamus, primary visual area, and higher-order visual area. BOLD responses increased slightly with pulse duration, were maximal at 3 - 5 Hz stimulation, and significantly decreased at 10 Hz, which were all consistent with previous neurophysiological findings. When the mice were awake, the BOLD fMRI response was faster in all active regions and stronger in the subcortical areas compared with the anesthesia condition. In the V1, the BOLD response was biphasic for 5 Hz stimulation and negative for 10 Hz stimulation under wakefulness, whereas prolonged positive BOLD responses were observed at both frequencies under anesthesia. Unexpected activation was detected in the extrastriate postrhinal area and non-visual subiculum complex under anesthesia, but not under wakefulness. Widespread positive BOLD activity under anesthesia likely results from the disinhibition and sensitization of excitatory neurons induced by ketamine. Overall, fMRI can be a viable tool for mapping brain-wide functional networks.