Mosaic and non-mosaic pcdh19 mutation leads to neuronal hyperexcitability in zebrafish

Epilepsy is one of the most common neurological disorders. The X-linked gene PCDH19 is associated with sporadic and familial epilepsy in humans, typically with early-onset clustering seizures and intellectual disability in females but not in so-called carrier males, suggesting that mosaic PCDH19 expression is required to produce epilepsy. To characterize the role of loss of PCDH19 function in epilepsy, we generated zebrafish with truncating pcdh19 variants. We observed hyperexcitability phenotypes in both mosaic and non-mosaic pcdh19+/- and -/- mutant larvae, indicating that Pcdh19 cellular mosaicism is not required for network hyperexcitability in zebrafish. Further, zebrafish with non-mosaic pcdh19 mutation display reduced numbers of inhibitory interneurons and transcriptional down-regulation of key inhibitory synapse components, suggesting a potential cellular basis for the observed hyperexcitability. Our findings in both mosaic and non-mosaic pcdh19 mutant zebrafish challenge the prevailing theory that mosaicism governs all PCDH19-related phenotypes and point to interneuron-mediated mechanisms underlying these phenotypes.

Inhibitory control in neuronal networks relies on the extracellular matrix integrity

Inhibitory control is essential for the regulation of neuronal network activity, where excitatory and inhibitory synapses can act synergistically, reciprocally, and antagonistically. Sustained excitation-inhibition (E-I) balance, therefore, relies on the orchestrated adjustment of excitatory and inhibitory synaptic strength. While growing evidence indicates that the brain’s extracellular matrix (ECM) is a crucial regulator of excitatory synapse plasticity, it remains unclear whether and how the ECM contributes to inhibitory control in neuronal networks. Here we studied the simultaneous changes in excitatory and inhibitory connectivity after ECM depletion. We demonstrate that the ECM supports the maintenance of E-I balance by retaining inhibitory connectivity. Quantification of synapses and super-resolution microscopy showed that depletion of the ECM in mature neuronal networks preferentially decreases the density of inhibitory synapses and the size of individual inhibitory postsynaptic scaffolds. The reduction of inhibitory synapse density is partially compensated by the homeostatically increasing synaptic strength via the reduction of presynaptic GABAB receptors, as indicated by patch-clamp measurements and GABAB receptor expression quantifications. However, both spiking and bursting activity in neuronal networks is increased after ECM depletion, as indicated by multi-electrode recordings. With computational modelling, we determined that ECM depletion reduces the inhibitory connectivity to an extent that the inhibitory synapse scaling does not fully compensate for the reduced inhibitory synapse density. Our results indicate that the brain’s ECM preserves the balanced state of neuronal networks by supporting inhibitory control via inhibitory synapse stabilization, which expands the current understanding of brain activity regulation. Graphic abstract

ErbB4 promotes inhibitory synapse formation by cell adhesion, independent of its kinase activity

The precise control of the nervous system function under the vitality of synapses is extremely critical. Efforts have been taken to explore the underlying cellular and molecular mechanisms for synapse formation. Cell adhesion molecules have been found important for synapse assembly in the brain. Many trans-adhesion complexes have been identified to modulate excitatory synapse formation. However, little is known about the synaptogenic mechanisms for inhibitory synapses. ErbB4 is a receptor tyrosine kinase enriched in interneurons. Here, we showed that overexpressing ErbB4 in HEK293T cells induced gephyrin or GABAAR α1 puncta in co-cultured primary hippocampal neurons. This induction of ErbB4 was independent of its kinase activity. K751M, a kinase-dead mutant of ErbB4, can also induce gephyrin or GABAAR α1 puncta in the co-culture system. We further constructed K751M knock-in mice and found that the homozygous were viable at birth and fertile without changes in gross brain structure. The number of interneurons and inhibitory synapses onto pyramidal neurons (PyNs) were comparable between K751M and wild-type mice but decreased in ErbB4-Null mice. Moreover, ErbB4 can interact in trans with Slitrk3, a transmembrane postsynaptic protein at inhibitory synapses, through the extracellular RLD domain of ErbB4. The deletion of RLD diminished the induction of gephyrin or GABAAR α1 puncta by ErbB4. Finally, disruption of ErbB4–Slitrk3 interaction through neutralization of Slitrk3 by secretable RLD decreased inhibitory synapses onto PyNs and impaired GABAergic transmission. These results identify that ErbB4, as a cell adhesion molecule, promotes inhibitory synapse formation onto PyNs by interacting with Slitrk3 and in a kinase-independent manner, providing an unexpected mechanism of ErbB4 in inhibitory synapse formation.

Noise Exposure Alters Glutamatergic and GABAergic Synaptic Connectivity in the Hippocampus and Its Relevance to Tinnitus

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.

Ste20-like kinase is critical for inhibitory synapse maintenance and its deficiency confers a developmental dendritopathy

SummaryThe size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.