Over the past decades, it has become increasingly clear that many neurodevelopmental disorders can be characterized by aberrations in the neuro-anatomical connectome of intermediary hubs. Yet,... Show moreOver the past decades, it has become increasingly clear that many neurodevelopmental disorders can be characterized by aberrations in the neuro-anatomical connectome of intermediary hubs. Yet, despite the advent in unidirectional transsynaptic tracing technologies, we are still lacking an efficient approach to identify individual neurons based on both their precise input and output relations, hampering our ability to elucidate the precise connectome in both the healthy and diseased condition. Here, we bridge this gap by combining anterograde transsynaptic- and retrograde (cATR) tracing in Ai14 reporter mice, using adeno-associated virus serotype 1 expressing Cre and cholera toxin subunit B as the anterograde and retrograde tracer, respectively. We have applied this innovative approach to selectively identify individual neurons in the brainstem that do not only receive input from one or more of the cerebellar nuclei (CN), but also project to the primary motor cortex (M1), the amygdala or the ventral tegmental area (VTA). Cells directly connecting CN to M1 were found mainly in the thalamus, while a large diversity of midbrain and brainstem areas connected the CN to the amygdala or VTA. Our data highlight that cATR allows for specific, yet brain-wide, identification of individual neurons that mediate information from a cerebellar nucleus to the cerebral cortex, amygdala or VTA via a disynaptic pathway. Given that the identified neurons in healthy subjects can be readily quantified, our data also form a solid foundation to make numerical comparisons with mouse mutants suffering from aberrations in their connectome due to a neurodevelopmental disorder. Show less
Kaiser, F.M.P.; Gruenbacher, S.; Oyaga, M.R.; Nio, E.; Jaritz, M.; Sun, Q.; ... ; Busslinger, M. 2022
The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic... Show moreThe genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome. Show less
Background: Epileptic (absence) seizures in the cerebral cortex can be stopped by pharmacological and optogenetic stimulation of the cerebellar nuclei (CN) neurons that innervate the thalamus.... Show moreBackground: Epileptic (absence) seizures in the cerebral cortex can be stopped by pharmacological and optogenetic stimulation of the cerebellar nuclei (CN) neurons that innervate the thalamus. However, it is unclear how such stimulation can modify underlying thalamo-cortical oscillations. Hypothesis: Here we tested whether rhythmic synchronized thalamo-cortical activity during absence seizures can be desynchronized by single-pulse optogenetic stimulation of CN neurons to stop seizure activity. Methods: We performed simultaneous thalamic single-cell and electrocorticographical recordings in awake tottering mice, a genetic model of absence epilepsy, to investigate the rhythmicity and synchronicity. Furthermore, we tested interictally the impact of single-pulse optogenetic CN stimulation on thalamic and cortical recordings. Results: We show that thalamic firing is highly rhythmic and synchronized with cortical spike-and-wave discharges during absence seizures and that this phase-locked activity can be desynchronized upon single-pulse optogenetic stimulation of CN neurons. Notably, this stimulation of CN neurons was more effective in stopping seizures than direct, focal stimulation of groups of afferents innervating the thalamus. During interictal periods, CN stimulation evoked reliable but heterogeneous responses in thalamic cells in that they could show an increase or decrease in firing rate at various latencies, bi-phasic responses with an initial excitatory and subsequent inhibitory response, or no response at all. Conclusion: Our data indicate that stimulation of CN neurons and their fibers in thalamus evokes differential effects in its downstream pathways and desynchronizes phase-locked thalamic neuronal firing during seizures, revealing a neurobiological mechanism that may explain how cerebellar stimulation can stop seizures. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND Show less
Genzel, L.; Adan, R.; Berns, A.; Beucken, J. van den; Blokland, A.; Boddeke, E.H.W.G.M.; ... ; Homberg, J.R. 2020
Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible,... Show moreRecently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternative methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and the various animal-free alternatives currently available do not come close to this complexity. In this Essay, we therefore argue that the use of animals is essential for the advancement of human and veterinary health. Show less
Bechakra, M.; Nieuwenhoff, M.D.; Rosmalen, J. van; Groeneveld, G.J.; Huygen, F.J.P.M.; Zeeuw, C.I. de; ... ; Jongen, J.L.M. 2020
Consistent associations between the severity of neuropathic pain and cutaneous innervation have not been described. We collected demographic and clinical data, McGill Pain Questionnaires (MPQ) and... Show moreConsistent associations between the severity of neuropathic pain and cutaneous innervation have not been described. We collected demographic and clinical data, McGill Pain Questionnaires (MPQ) and skin biopsies processed for PGP9.5 and CGRP immunohistochemistry from patients with bortezomib-induced peripheral neuropathy (BiPN; n = 22), painful diabetic neuropathy (PDN; n = 16), chronic idiopathic axonal polyneuropathy (CIAP; n = 16) and 17 age -matched healthy volunteers. Duration of neuropathic symptoms was significantly shorter in patients with BiPN in comparison with PDN and CLAP patients. BiPN was characterized by a significant increase in epidermal axonal swellings and upper dermis nerve fiber densities (UDNFD) and a decrease in subepidermal nerve fiber densities (SENFD) of PGP9.5-positive fibers and of PGP9.5 containing structures that did not show CGRP labeling, presumably non-peptidergic fibers. In PDN and CIAP patients, intraepidermal nerve fiber densities (IENFD) and SENFD of PGP9.5-positive and of non-peptidergic fibers were decreased in comparison with healthy volunteers. Significant unadjusted associations between LENFD and SENFD of CGRP-positive, i.e. peptidergic, fibers and the MPQ sensory-discriminative, as well as between UDNFD of PGP9.5-positive fibers and the MPQ evaluative/affective component of neuropathic pain, were found in BiPN and CIAP patients. No significant associations were found in PDN patients. Cutaneous innervation changes in BiPN confirm characteristic features of early, whereas those in CIAP and PDN are in line with late forms of neuropathic pathology. Our results allude to a distinct role for non-peptidergic nociceptors in BiPN and CIAP patients. The lack of significant associations in PDN may be caused by mixed ischemic and purely neuropathic pain pathology. Show less
Esbroeck, A.C.M. van; Janssen, A.P.A.; Cognetta III, A.B.; Ogasawara, D.; Shpak, G.; Kroeg, M. van der; ... ; Stelt, M. van der 2017
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although... Show moreA recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system. Show less
Objective: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is... Show moreObjective: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. Methods: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Results: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. Interpretation: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema. ANN NEUROL 2010;67:85-98 Show less
