Moving From Transactional to Relational: How Funders Can Work in Partnership With Black, Indigenous and People of Color Communities.

BACKGROUND: The Racial Equity Coalition (REC) formed to address persistent educational disparities. The coalition was composed of 14 Black, Indigenous and People of Color (BIPOC) organizations that provide culturally integrative youth services. OBJECTIVES: REC, with support from United Way of King County, engaged in participatory research to identify commonalities and shared struggles to inform collective action. Participatory research aligns with REC's commitment to equitable participatory processes. This article focuses on REC's experiences with funders. The objective was to understand what creates positive and challenging experiences with funders, and to identify recommendations for funders to become more culturally responsive. METHODS: A research committee was formed including representatives of nine REC organizations and United Way of King County staff. The committee conducted interviews with each of the 14 REC organizations and conducted thematic analysis of interview transcripts. Through participatory analysis, the committee drafted narratives that were further refined through a series of research retreats attended by all REC organizations. RESULTS: Recommendations were to incentivize collaboration, listen to communities to create culturally responsive definitions of success and measurement strategies, arrive at mutually agreed upon approaches with organizations, honor the connections BIPOC organizations have with their communities, and provide unrestricted funding to allow BIPOC organizations greater agency. CONCLUSIONS: A major challenge for BIPOC organizations is navigating White dominant culture that too often shows up in funding requirements. Having to fit dominant culture standards stifles BIPOC organizations' abilities to meet community needs and the responsiveness of their approaches. REC identified recommendations for funders to be more culturally responsive and community centered.

Damaging de novo missense variants in EEF1A2 lead to a developmental and degenerative epileptic-dyskinetic encephalopathy.

Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein-damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic-dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.

Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells.

Mitochondria play a prominent role in mechanosensory hair cell damage and death. Although hair cells are thought to be energetically demanding cells, how mitochondria respond to these demands and how this might relate to cell death is largely unexplored. Using genetically encoded indicators, we found that mitochondrial calcium flux and oxidation are regulated by mechanotransduction and demonstrate that hair cell activity has both acute and long-term consequences on mitochondrial function. We tested whether variation in mitochondrial activity reflected differences in the vulnerability of hair cells to the toxic drug neomycin. We observed that susceptibility did not correspond to the acute level of mitochondrial activity but rather to the cumulative history of that activity.

Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse.

We report functional and structural evidence for GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss.SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca2+-permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca2+ accumulation and swelling that can be prevented by blocking CP-AMPARs. We demonstrate that CP-AMPARs mediate transmission at this first-order sensory synapse and that limiting Ca2+ accumulation in the terminal may protect against hearing loss.

GABAB receptors in maintenance of neocortical circuit function.

Activation of metabotropic GABAB receptors (GABABRs) enhances tonic GABA current and substantially increases the frequency of spontaneous seizures. Despite the and pro-epileptic consequences of GABABR activation, mice lacking functional GABAB receptors (GABAB1R KO mice) exhibit clonic and rare absence seizures. To examine these mutant mice further, we recorded excitatory and inhibitory synaptic inputs and tonic mutant GABA currents from Layer 2 neocortical pyramidal neurons of GABAB1R WT and KO mice (P30-40). Tonic current was increased while the frequency of synaptic inputs was unchanged in KO mice relative to WT littermates. The neocortical laminar distribution of interneuron subtypes derived from the medial ganglionic eminence (MGE) was also not statistically different in KO mice relative to WT while the number of calretinin-positive, caudal GE-derived cells in Layer 1 was reduced. Transplantation of MGE progenitors obtained from KO mice lacking functional GABAB1R did not increase tonic inhibition in the host brain above that of media-injected controls. Taken together, these results suggest a complex role for GABAB receptors in mediating neocortical circuit function.

Neocortical integration of transplanted GABA progenitor cells from wild type and GABA(B) receptor knockout mouse donors.

Most cortical interneurons originate in a region of the embryonic subpallium called the medial ganglionic eminence (MGE). When MGE cells are transplanted into cerebral cortex, these progenitors migrate extensively and differentiate into functional inhibitory neurons. Although MGE progenitors have therapeutic potential following transplantation, it is unknown precisely how these cells distribute within neocortical lamina of the recipient brain. Here we transplanted mouse embryonic day 12.5 MGE progenitors into postnatal neocortex and evaluated laminar distribution of interneuron subtypes using double- and triple-label immunohistochemistry. Studies were performed using wild type (WT) or donor mice lacking a metabotropic GABA(B) receptor subunit (GABA(B1)R KO). MGE-derived neurons from WT and GABA(B1)R KO mice preferentially and densely distributed in neocortical layers 2/3, 5 and 6. As expected, MGE-derived neurons differentiated into parvalbumin+ and somatostatin+ interneurons within these neocortical lamina. Our findings provide insights into the anatomical integration of MGE-derived interneurons following transplantation.

ALLN rescues an in vitro excitatory synaptic transmission deficit in Lis1 mutant mice.

LIS1 gene mutations lead to a rare neurological disorder, classical lissencephaly, characterized by brain malformations, mental retardation, seizures, and premature death. Mice heterozygous for Lis1 (Lis1(+/-)) exhibit cortical malformations, defects in neuronal migration, increased glutamate-mediated synaptic transmission, and spontaneous electrographic seizures. Recent work demonstrated that in utero treatment of Lis1(+/-) mutant dams with ALLN, a calpain inhibitor, partially rescues neuronal migration defects in the offspring. Given the challenges of in utero drug administration, we examined the therapeutic potential of ALLN on postnatal lissencephalic cells. Voltage- and current-clamp studies were performed with acute hippocampal slices obtained from Lis1 mutant mice and age-matched littermate control mice. Specifically, we determined whether postnatal ALLN treatment can reverse excitatory synaptic transmission deficits, namely, an increase in spontaneous and miniature excitatory postsynaptic current (EPSC) frequency, on CA1 pyramidal neurons observed in tissue slices from Lis1(+/-) mice. We found that acute application of ALLN restored spontaneous and miniature EPSC frequencies to wild-type levels without affecting inhibitory postsynaptic synaptic current. Furthermore, Western blot analysis of protein expression, including proteins involved in excitatory synaptic transmission, demonstrated that ALLN blocks the cleavage of the calpain substrate αII-spectrin but does not rescue Lis1 protein levels in Lis1(+/-) mutants.

Intrinsically determined cell death of developing cortical interneurons.

Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis, is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo, in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.

The promise of an interneuron-based cell therapy for epilepsy.

Of the nearly 3 million Americans diagnosed with epilepsy, approximately 30% are unresponsive to current medications. Recent data has shown that early postnatal transplantation of interneuronal precursor cells increases GABAergic inhibition in the host brain and dramatically suppresses seizure activity in epileptic mice. In this review, we will highlight findings from seizure-prone mice and humans that demonstrate the link between dysfunctional GABAergic inhibition and hyperexcitability. In particular, we will focus on rodent models of temporal lobe epilepsy, the most common and difficult to treat form of the disease, and interneuronopathies, an emerging classification. A wealth of literature showing a causal link between reduced GABA-mediated inhibition and seizures has directed our efforts to recover the loss of inhibition via transplantation of interneuronal precursors. Numerous related studies have explored the anticonvulsant potential of cell grafts derived from a variety of brain regions, yet the mechanism underlying the effect of such heterogeneous cell transplants is unknown. In discussing our recent findings and placing them in context with what is known about epilepsy, and how related transplant approaches have progressed, we hope to initiate a frank discussion of the best path toward the translation of this approach to patients with intractable forms of epilepsy.

Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells.

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl(-) reversal potential (E(Cl)). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA(A) receptors (GABA(A) Rs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2-4] then decreases dramatically by the second postnatal week (P7-10 and P30-40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA(A) R α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 µM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E(Cl) recorded from P2-5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.

Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.

Epilepsy, a disease characterized by abnormal brain activity, is a disabling and potentially life-threatening condition for nearly 1% of the world population. Unfortunately, modulation of brain excitability using available antiepileptic drugs can have serious side effects, especially in the developing brain, and some patients can only be improved by surgical removal of brain regions containing the seizure focus. Here, we show that bilateral transplantation of precursor cells from the embryonic medial ganglionic eminence (MGE) into early postnatal neocortex generates mature GABAergic interneurons in the host brain. In mice receiving MGE cell grafts, GABA-mediated synaptic and extrasynaptic inhibition onto host brain pyramidal neurons is significantly increased. Bilateral MGE cell grafts in epileptic mice lacking a Shaker-like potassium channel (a gene mutated in one form of human epilepsy) resulted in significant reductions in the duration and frequency of spontaneous electrographic seizures. Our findings suggest that MGE-derived interneurons could be used to ameliorate abnormal excitability and possibly act as an effective strategy in the treatment of epilepsy.

Developmental effects of ketamine on inspiratory hypoglossal nerve activity studied in vivo and in vitro.

The effects of the anesthetic ketamine on properties of inspiratory bursts (I-bursts) in mouse hypoglossal nerve activity were studied in vivo and in vitro. In urethane anesthetized mice we observed rhythmic I-phase activity in only one of eight pups at P9 days. In contrast in older mice rhythmic I-phase hypoglossal activity was almost always observed. Ketamine caused a reduction in I-burst frequency and an increase in peak integrated hypoglossal nerve activity in all three age groups studied (P10-P13, P15-P20 and adult mice). In these mice I-phase oscillations, due to hypoglossal motoneurons firing clusters of action potentials at a particular frequency, were observed in control and after ketamine. Ketamine did not change the frequency of the dominant spectral peak determined from power spectra examined from 0 to 200 Hz. The effects of ketamine were also studied in vitro in the mouse rhythmic medullary slice preparation. Ketamine reduced hypoglossal I-burst frequency and I-burst peak integrated amplitude. Oscillations were observed in I-phase activity, and as in the in vivo studies ketamine did not shift the dominant spectral peak frequency. These results demonstrate that in vivo and in vitro ketamine results in significant changes in I-burst frequency and peak integrated hypoglossal nerve activity, but changes in the oscillation frequency are minimal.

Inhibitory synaptic transmission governs inspiratory motoneuron synchronization.

Neurons within the intact respiratory network produce bursts of action potentials that cause inspiration or expiration. Within inspiratory bursts, activity is synchronized on a shorter timescale to generate clusters of action potentials that occur in a set frequency range and are called synchronous oscillations. We investigated how GABA and glycine modulate synchronous oscillations and respiratory rhythm during postnatal development. We recorded inspiratory activity from hypoglossal nerves using the in vitro rhythmically active mouse medullary slice preparation from P0-P11 mice. Average oscillation frequency increased with postnatal development, from 17 +/- 12 Hz in P0-P6 mice (n = 15) to 38 +/- 7 Hz in P7-P11 mice (n = 37) (P < 0.0001). Bath application of GABAA and GlyR antagonists significantly reduced oscillation power in neonates (P0-P6) and juveniles (P7-P10) and increased peak integrated activity in both age groups. To test whether elevating slice excitability is sufficient to reduce oscillation power, Substance P was bath applied alone. Substance P, although increasing peak integrated activity, had no significant effect on oscillation power. Prolonging the time course of GABAergic synaptic currents with zolpidem decreased the median oscillation frequency in P9-P10 mouse slices. These data demonstrate that oscillation frequency increases with postnatal development and that both GABAergic and glycinergic transmission contribute to synchronization of activity. Further, the time course of synaptic GABAergic currents is a determinant of oscillation frequency.

GABA(B) modulation of GABA(A) and glycine receptor-mediated synaptic currents in hypoglossal motoneurons.

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.

Differential effects of ethanol on GABA(A) and glycine receptor-mediated synaptic currents in brain stem motoneurons.

Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9-13) are more sensitive to ethanol than neonate HMs (P1-3). We have now extended our previous study to investigate ethanol modulation of synaptic GABA(A) receptors (GABA(A)Rs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 +/- 3%, P < 0.05) and juvenile (16 +/- 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 +/- 17%, P < 0.05) and juvenile (40 +/- 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 +/- 3%, P < 0.0001) and juvenile (41 +/- 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABA(A) receptors and that 100 mM ethanol has the opposite effect on GABA(A)R-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.