Human brain mapping using co-registered fUS, fMRI and ESM during awake brain surgeries: A proof-of-concept study.

Accurate, depth-resolved functional imaging is key in both understanding and treatment of the human brain. A new sonography-based imaging technique named functional Ultrasound (fUS) uniquely combines high sensitivity with submillimeter-subsecond spatiotemporal resolution available in large fields-of-view. In this proof-of-concept study we show that: (A) fUS reveals the same eloquent regions as found by fMRI while concomitantly visualizing in-vivo microvascular morphology underlying these functional hemodynamics and (B) fUS-based functional maps are confirmed by Electrocortical Stimulation Mapping (ESM), the current gold-standard in awake neurosurgical practice. This unique cross-modality experiment was performed using motor, visual and language-related functional tasks in patients undergoing awake brain tumor resection. The current work serves as an important milestone towards further maturity of fUS as well as a novel avenue to increase our understanding of hemodynamics-based functional brain imaging.

Impact of enriched environment on motor performance and learning in mice.

Neuroscience heavily relies on animal welfare in laboratory rodents as it can significantly affect brain development, cognitive function and memory formation. Unfortunately, laboratory animals are often raised in artificial environments devoid of physical and social stimuli, potentially leading to biased outcomes in behavioural assays. To assess this effect, we examined the impact of social and physical cage enrichment on various forms of motor coordination. Our findings indicate that while enriched-housed animals did not exhibit faster learning in eyeblink conditioning, the peak timing of their conditioned responses was slightly, but significantly, improved. Additionally, enriched-housed animals outperformed animals that were housed in standard conditions in the accelerating rotarod and ErasmusLadder test. In contrast, we found no significant effect of enrichment on the balance beam and grip strength test. Overall, our data suggest that an enriched environment can improve motor performance and motor learning under challenging and/or novel circumstances, possibly reflecting an altered state of anxiety.

A retinal origin of nystagmus-a perspective.

Congenital nystagmus is a condition where the eyes of patients oscillate, mostly horizontally, with a frequency of between 2 and 10 Hz. Historically, nystagmus is believed to be caused by a maladaptation of the oculomotor system and is thus considered a disease of the brain stem. However, we have recently shown that congenital nystagmus associated with congenital stationary night blindness is caused by synchronously oscillating retinal ganglion cells. In this perspective article, we discuss how some details of nystagmus can be accounted for by the retinal mechanism we propose.

Accessible and reliable neurometric testing in humans using a smartphone platform.

Tests of human brain circuit function typically require fixed equipment in lab environments. We have developed a smartphone-based platform for neurometric testing. This platform, which uses AI models like computer vision, is optimized for at-home use and produces reproducible, robust results on a battery of tests, including eyeblink conditioning, prepulse inhibition of acoustic startle response, and startle habituation. This approach provides a scalable, universal resource for quantitative assays of central nervous system function.

Stimulus Generalization in Mice during Pavlovian Eyeblink Conditioning.

Here, we investigate stimulus generalization in a cerebellar learning paradigm, called eyeblink conditioning. Mice were conditioned to close their eyes in response to a 10-kHz tone by repeatedly pairing this tone with an air puff to the eye 250 ms after tone onset. After 10 consecutive days of training, when mice showed reliable conditioned eyelid responses to the 10-kHz tone, we started to expose them to tones with other frequencies, ranging from 2 to 20 kHz. We found that mice had a strong generalization gradient, whereby the probability and amplitude of conditioned eyelid responses gradually decreases depending on the dissimilarity with the 10-kHz tone. Tones with frequencies closest to 10 kHz evoked the most and largest conditioned eyelid responses and each step away from the 10-kHz tone resulted in fewer and smaller conditioned responses (CRs). In addition, we found that tones with lower frequencies resulted in CRs that peaked earlier after tone onset compared with those to tones with higher frequencies. Together, our data show prominent generalization patterns in cerebellar learning. Since the known function of cerebellum is rapidly expanding from pure motor control to domains that include cognition, reward-learning, fear-learning, social function, and even addiction, our data imply generalization controlled by cerebellum in all these domains.

Olivocerebellar control of movement symmetry.

Coordination of bilateral movements is essential for a large variety of animal behaviors. The olivocerebellar system is critical for the control of movement, but its role in bilateral coordination has yet to be elucidated. Here, we examined whether Purkinje cells encode and influence synchronicity of left-right whisker movements. We found that complex spike activity is correlated with a prominent left-right symmetry of spontaneous whisker movements within parts, but not all, of Crus1 and Crus2. Optogenetic stimulation of climbing fibers in the areas with high and low correlations resulted in symmetric and asymmetric whisker movements, respectively. Moreover, when simple spike frequency prior to the complex spike was higher, the complex spike-related symmetric whisker protractions were larger. This finding alludes to a role for rebound activity in the cerebellar nuclei, which indeed turned out to be enhanced during symmetric protractions. Tracer injections suggest that regions associated with symmetric whisker movements are anatomically connected to the contralateral cerebellar hemisphere. Together, these data point toward the existence of modules on both sides of the cerebellar cortex that can differentially promote or reduce the symmetry of left and right movements in a context-dependent fashion.

Cerebellum-dependent associative learning is not impaired in a mouse model of neurofibromatosis type 1.

Individuals with Neurofibromatosis type 1 (NF1) experience a high degree of motor problems. The cerebellum plays a pivotal role in motor functioning and the NF1 gene is highly expressed in cerebellar Purkinje cells. However, it is not well understood to what extent NF1 affects cerebellar functioning and how this relates to NF1 motor functioning. Therefore, we subjected global Nf1+/- mice to a cerebellum-dependent associative learning task, called Pavlovian eyeblink conditioning. Additionally, we assessed general motor function and muscle strength in Nf1+/- mice. To our surprise, we found that Nf1+/- mice showed a moderately increased learning rate of conditioned eyeblink responses, as well as improved accuracy in the adaptive timing of the eyeblink responses. Locomotion, balance, general motor function, and muscle strength were not affected in Nf1+/- mice. Together, our results support the view that cerebellar function in Nf1+/- mice is unimpaired.

Increased noise level of purkinje cell activities minimizes impact of their modulation during sensorimotor control.

While firing rate is well established as a relevant parameter for encoding information exchanged between neurons, the significance of other parameters is more conjectural. Here, we show that regularity of neuronal spike activities affects sensorimotor processing in tottering mutants, which suffer from a mutation in P/Q-type voltage-gated calcium channels. While the modulation amplitude of the simple spike firing rate of their floccular Purkinje cells during optokinetic stimulation is indistinguishable from that of wild-types, the regularity of their firing is markedly disrupted. The gain and phase values of tottering's compensatory eye movements are indistinguishable from those of flocculectomized wild-types or from totterings with the flocculus treated with P/Q-type calcium channel blockers. Moreover, normal eye movements can be evoked in tottering when the flocculus is electrically stimulated with regular spike trains mimicking the firing pattern of normal simple spikes. This study demonstrates the importance of regularity of firing in Purkinje cells for neuronal information processing.

Deletion of FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar eyelid conditioning in Fragile X syndrome.

Absence of functional FMRP causes Fragile X syndrome. Abnormalities in synaptic processes in the cerebral cortex and hippocampus contribute to cognitive deficits in Fragile X patients. So far, the potential roles of cerebellar deficits have not been investigated. Here, we demonstrate that both global and Purkinje cell-specific knockouts of Fmr1 show deficits in classical delay eye-blink conditioning in that the percentage of conditioned responses as well as their peak amplitude and peak velocity are reduced. Purkinje cells of these mice show elongated spines and enhanced LTD induction at the parallel fiber synapses that innervate these spines. Moreover, Fragile X patients display the same cerebellar deficits in eye-blink conditioning as the mutant mice. These data indicate that a lack of FMRP leads to cerebellar deficits at both the cellular and behavioral levels and raise the possibility that cerebellar dysfunctions can contribute to motor learning deficits in Fragile X patients.

Conditioned climbing fiber responses in cerebellar cortex and nuclei.

The eyeblink conditioning paradigm captures an elementary form of associative learning in a neural circuitry that is understood to an extraordinary degree. Cerebellar cortical Purkinje cell simple spike suppression is widely regarded as the main process underlying conditioned responses (CRs), leading to disinhibition of neurons in the cerebellar nuclei that innervate eyelid muscles downstream. However, recent work highlights the addition of a conditioned Purkinje cell complex spike response, which at the level of the interposed nucleus seems to translate to a transient spike suppression that can be followed by a rapid spike facilitation. Here, we review the characteristics of these responses at the cerebellar cortical and nuclear level, and discuss possible origins and functions.

CLIP-115, a novel brain-specific cytoplasmic linker protein, mediates the localization of dendritic lamellar bodies.

Intracellular localization of organelles may depend in part on specific cytoplasmic linker proteins (CLIPs) that link membranous organelles to microtubules. Here, we characterize rat cDNAs encoding a novel, brain-specific CLIP of 115 kDa. This protein contains two N-terminal microtubule-binding domains and a long coiled-coil region; it binds to microtubules and is homologous to CLIP-170, a protein mediating the binding of endosomes to microtubules. CLIP-115 is enriched in the dendritic lamellar body (DLB), a recently discovered organelle predominantly present in bulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. Local microtubule depolymerization leads to a temporary reduction of DLBs. These results suggest that CLIP-115 operates in the control of brain-specific organelle translocations.

Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the vestibulo-ocular reflex.

Cerebellar long-term depression (LTD) is a model system for neuronal information storage that has an absolute requirement for activation of protein kinase C (PKC). It has been claimed to underlie several forms of cerebellar motor learning. Previous studies using various knockout mice (mGluR1, GluRdelta2, glial fibrillary acidic protein) have supported this claim; however, this work has suffered from the limitations that the knockout technique lacks anatomical specificity and that functional compensation can occur via similar gene family members. To overcome these limitations, a transgenic mouse (called L7-PKCI) has been produced in which the pseudosubstrate PKC inhibitor, PKC[19-31], was selectively expressed in Purkinje cells under the control of the pcp-2(L7) gene promoter. Cultured Purkinje cells prepared from heterozygous or homozygous L7-PKCI embryos showed a complete blockade of LTD induction. In addition, the compensatory eye movements of L7-PKCI mice were recorded during vestibular and visual stimulation. Whereas the absolute gain, phase, and latency values of the vestibulo-ocular reflex and optokinetic reflex of the L7-PKCI mice were normal, their ability to adapt their vestibulo-ocular reflex gain during visuo-vestibular training was absent. These data strongly support the hypothesis that activation of PKC in the Purkinje cell is necessary for cerebellar LTD induction, and that cerebellar LTD is required for a particular form of motor learning, adaptation of the vestibulo-ocular reflex.

Tragedy of conducting a clinical trial; generic alert system needed.

Stopping a clinical trial without reaching the final objective is not the ideal outcome any researcher wants; sometimes ceasing is inevitable. Due to marginal inclusion of patients we were forced to cease our randomized clinical trial on the effectiveness of proprioceptive training on the development of chronic whiplash complaints a year after the start. Although incidence figures demonstrate that recruitment of the planned number of whiplash patients would be easily feasible, we were unable to enroll the amount of subjects. Several motives can be proposed that would have prevented this obliged halting from happening. Other studies also report impracticability of the planned number of whiplash injury patients.

Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules.

Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.

Microcircuitry and function of the inferior olive.

The inferior olive, which provides the climbing fibers to Purkinje cells in the cerebellar cortex, has been implicated in various functions, such as learning and timing of movements, and comparing intended with achieved movements. For example, climbing-fiber activity could transmit error signals during eye-blink conditioning or adaptation of the vestibulo-ocular reflex, or it could carry motor command signals beating on the rhythm of the oscillating and synchronous firing of ensembles of olivary neurons, or both. In this review, we approach the controversial issue of olivocerebellar function from the perspective of the unique organization of the microcircuitry of the olivary neuropil. The characteristic glomeruli are formed by a core of long dendritic or axonal spines, each of which is innervated by both an inhibitory terminal derived from the hindbrain and an excitatory terminal derived from either an ascending or descending input. The dendritic spines, which originate from dendrites with varicosities carrying dendritic lamellar bodies, are coupled by gap junctions. By drawing a comparison with a computational model by Segev and Rall,which might be applicable to the typical olivary spine with its unique morphological features and combined excitatory and inhibitory input, we propose that the microcircuitry of the inferior olive is capable of functioning both in motor learning and motor timing, but does not directly compare intended with achieved movements.

GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei.

The GATA-3 transcription factor shows a specific and restricted expression pattern in the developing and adult mouse brain. In the present study we investigated the role of GATA-3 in the caudal raphe system, which is known to operate as a modulator of motor activity. We demonstrate that virtually all neurons in the caudal raphe nuclei that express GATA-3 also produce serotonin. Absence of GATA-3, as analyzed in chimeric -/- mice, affects the cytoarchitecture of serotonergic neurons in the caudal raphe nuclei. As a result the chimeras show a serious defect in their locomotor performance on a rotating rod. In sum, we conclude that GATA-3 plays a major role in the development of the serotonergic neurons of the caudal raphe nuclei, and that it is crucial for their role in locomotion.

Expression of protein kinase C inhibitor blocks cerebellar long-term depression without affecting Purkinje cell excitability in alert mice.

A longstanding but still controversial hypothesis is that long-term depression (LTD) of parallel fiber-Purkinje cell synapses in the cerebellum embodies part of the neuronal information storage required for associative motor learning. Transgenic mice in which LTD is blocked by Purkinje cell-specific inhibition of protein kinase C (PKC) (L7-PKCI mutants) do indeed show impaired adaptation of their vestibulo-ocular reflex, whereas the dynamics of their eye movement performance are unaffected. However, because L7-PKCI mutants have a persistent multiple climbing fiber innervation at least until 35 d of age and because the baseline discharge of the Purkinje cells in the L7-PKCI mutants is unknown, factors other than a blockage of LTD induction itself may underlie their impaired motor learning. We therefore investigated the spontaneous discharge of Purkinje cells in alert adult L7-PKCI mice as well as their multiple climbing fiber innervation beyond the age of 3 months. We found that the simple spike and complex spike-firing properties (such as mean firing rate, interspike interval, and spike count variability), oscillations, and climbing fiber pause in the L7-PKCI mutants were indistinguishable from those in their wild-type littermates. In addition, we found that multiple climbing fiber innervation does not occur in cerebellar slices obtained from 3- to 6-month-old mutants. These data indicate (1) that neither PKC inhibition nor the subsequent blockage of LTD induction disturbs the spontaneous discharge of Purkinje cells in alert mice, (2) that Purkinje cell-specific inhibition of PKC detains rather than prevents the developmental conversion from multiple to mono-innervation of Purkinje cells by climbing fibers, and (3) that as a consequence the impaired motor learning as observed in older adult L7-PKCI mutants cannot be attributable either to a disturbance in the baseline simple spike and complex spike activities of their Purkinje cells or to a persistent multiple climbing fiber innervation. We conclude that cerebellar LTD is probably one of the major mechanisms underlying motor learning, but that deficits in LTD induction and motor learning as observed in the L7-PKCI mutants may only be reflected in differences of the Purkinje cell signals during and/or directly after training.

Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis.

The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.

Gap junctions synchronize synaptic input rather than spike output of olivary neurons.

Electronic coupling in the inferior olive is supposed to underlie the synchrony of complex spike activities of Purkinje cells in the cerebellar cortex. Here we show a computational model which suggests that the olivary gap junctions may synchronize the input rather than the neuronal output. As such, coupling may influence the absolute moment in time of the complex spike activity rather than their synchrony.

The cervico-ocular reflex is increased in whiplash injury patients.

Whiplash-associated disorders (WAD) are a major problem in the Western world, which put a formidable financial burden on modern society and which evoke an emerging debate on the true nature of their origin. To date there is no generally accepted test that allows us to diagnose WAD objectively. Because whiplash injury causes dysfunction of proprioception in the neck, we investigated the characteristics of the cervico-ocular reflex (COR) of presumptive WAD patients. These patients and age-matched healthy controls were rotated at different stimulus peak velocities in the dark while their head was fixed in space. The gain values of the COR were significantly increased in the patient population at a wide range of stimulus peak velocities with maximum difference at the lower frequencies (p = 0.037, ANOVA). Hence, although larger numbers of patients should be measured, the COR gain appears to be a parameter that may permit an objective diagnosis of WAD.