A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology.

ABSTRACT: Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. We present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. Osteoarthritis-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell and single action potential resolution and a throughput of up to 500,000 neurons per day. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology. We screened ∼3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including ion channel modulators and other mechanisms including MEK inhibitors and tyrosine kinase modulators. Our results suggest that the Raf-MEK-ERK axis in DRG neurons may integrate the inputs from multiple upstream inflammatory mediators found in osteoarthritis patient joints, and MAPK pathway activation in DRG neurons may contribute to chronic pain in patients with osteoarthritis.

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models.

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

Chronic stress induces cell type-selective transcriptomic and electrophysiological changes in the bed nucleus of the stria terminalis.

Distinct regions and cell types in the anterolateral group of the bed nucleus of the stria terminalis (BNSTALG) act to modulate anxiety in opposing ways. A history of chronic stress increases anxiety-like behavior with lasting electrophysiological effects on the BNSTALG. However, the opposing circuits within the BNSTALG suggest that stress may have differential effects on the individual cell types that comprise these circuits to shift the balance to favor anxiogenesis. Yet, the effects of stress are generally examined by treating all neurons within a particular region of the BNST as a homogenoeus population. We used patch-clamp electrophysiology and single-cell quantitative reverse transcriptase PCR (scRT-PCR) to determine how chronic shock stress (CSS) affects electrophysiological and neurochemical properties of Type I, Type II, and Type III neurons in the BNSTALG. We report that CSS resulted in changes in the input resistance, time constant, action potential waveform, and firing rate of Type III but not Type I or II neurons. Additionally, only the Type III neurons exhibited an increase in Crf mRNA and a decrease in striatal-enriched protein tyrosine phosphatase (Ptpn5) mRNA after CSS. In contrast, only non-Type III cells showed a reduction in calcium-permeable AMPA receptor (CP-AMPAR) current and changes in mRNA expression of genes encoding AMPA receptor subunits after CSS. Importantly, none of the effects of CSS observed were seen in all cell types. Our results suggest that Type III neurons play a unique role in the BNSTALG circuit and represent a population of CRF neurons particularly sensitive to chronic stress.

Dynamic corticostriatal activity biases social bonding in monogamous female prairie voles.

Adult pair bonding involves dramatic changes in the perception and valuation of another individual. One key change is that partners come to reliably activate the brain's reward system, although the precise neural mechanisms by which partners become rewarding during sociosexual interactions leading to a bond remain unclear. Here we show, using a prairie vole (Microtus ochrogaster) model of social bonding, how a functional circuit from the medial prefrontal cortex to nucleus accumbens is dynamically modulated to enhance females' affiliative behaviour towards a partner. Individual variation in the strength of this functional connectivity, particularly after the first mating encounter, predicts how quickly animals begin affiliative huddling with their partner. Rhythmically activating this circuit in a social context without mating biases later preference towards a partner, indicating that this circuit's activity is not just correlated with how quickly animals become affiliative but causally accelerates it. These results provide the first dynamic view of corticostriatal activity during bond formation, revealing how social interactions can recruit brain reward systems to drive changes in affiliative behaviour.

Morphology and dendritic maturation of developing principal neurons in the rat basolateral amygdala.

The basolateral nucleus of the amygdala (BLA) assigns emotional valence to sensory stimuli, and many amygdala-dependent behaviors undergo marked development during postnatal life. We recently showed principal neurons in the rat BLA undergo dramatic changes to their electrophysiological properties during the first postnatal month, but no study to date has thoroughly characterized changes to morphology or gene expression that may underlie the functional development of this neuronal population. We addressed this knowledge gap with reconstructions of biocytin-filled principal neurons in the rat BLA at postnatal days 7 (P7), 14, 21, 28, and 60. BLA principal neurons underwent a number of morphological changes, including a twofold increase in soma volume from P7 to P21. Dendritic arbors expanded significantly during the first postnatal month and achieved a mature distribution around P28, in terms of total dendritic length and distance from soma. The number of primary dendrites and branch points were consistent with age, but branch points were found farther from the soma in older animals. Dendrites of BLA principal neurons at P7 had few spines, and spine density increased nearly fivefold by P21. Given the concurrent increase in dendritic material, P60 neurons had approximately 17 times as many total spines as P7 neurons. Together, these developmental transitions in BLA principal neuron morphology help explain a number of concomitant electrophysiological changes during a critical period in amygdala development.

Prenatal stress, regardless of concurrent escitalopram treatment, alters behavior and amygdala gene expression of adolescent female rats.

Depression during pregnancy has been linked to in utero stress and is associated with long-lasting symptoms in offspring, including anxiety, helplessness, attentional deficits, and social withdrawal. Depression is diagnosed in 10-20% of expectant mothers, but the impact of antidepressant treatment on offspring development is not well documented, particularly for females. Here, we used a prenatal stress model of maternal depression to test the hypothesis that in utero antidepressant treatment could mitigate the effects of prenatal stress. We also investigated the effects of prenatal stress and antidepressant treatment on gene expression related to GABAergic and serotonergic neurotransmission in the amygdala, which may underlie behavioral effects of prenatal stress. Nulliparous female rats were implanted with osmotic minipumps delivering clinically-relevant concentrations of escitalopram and mated. Pregnant dams were exposed to 12 days of mixed-modality stressors, and offspring were behaviorally assessed in adolescence (postnatal day 28) and adulthood (beyond day 90) to determine the extent of behavioral change. We found that in utero stress exposure, regardless of escitalopram treatment, increased anxiety-like behavior in adolescent females and profoundly influenced amygdala expression of the chloride transporters KCC2 and NKCC1, which regulate GABAergic function. In contrast, prenatal escitalopram exposure alone elevated amygdala expression of 5-HT1A receptors. In adulthood, anxiety-like behavior returned to baseline and gene expression effects in the amygdala abated, whereas deficits emerged in novel object recognition for rats exposed to stress during gestation. These findings suggest prenatal stress causes age-dependent deficits in anxiety-like behavior and amygdala function in female offspring, regardless of antidepressant exposure.

Postnatal maturation of GABAergic transmission in the rat basolateral amygdala.

Many psychiatric disorders, including anxiety and autism spectrum disorders, have early ages of onset and high incidence in juveniles. To better treat and prevent these disorders, it is important to first understand normal development of brain circuits that process emotion. Healthy and maladaptive emotional processing involve the basolateral amygdala (BLA), dysfunction of which has been implicated in numerous psychiatric disorders. Normal function of the adult BLA relies on a fine balance of glutamatergic excitation and GABAergic inhibition. Elsewhere in the brain GABAergic transmission changes throughout development, but little is known about the maturation of GABAergic transmission in the BLA. Here we used whole cell patch-clamp recording and single-cell RT-PCR to study GABAergic transmission in rat BLA principal neurons at postnatal day (P)7, P14, P21, P28, and P35. GABAA currents exhibited a significant twofold reduction in rise time and nearly 25% reduction in decay time constant between P7 and P28. This corresponded with a shift in expression of GABAA receptor subunit mRNA from the α2- to the α1-subunit. The reversal potential for GABAA receptors transitioned from depolarizing to hyperpolarizing with age, from around -55 mV at P7 to -70 mV by P21. There was a corresponding shift in expression of opposing chloride pumps that influence the reversal, from NKCC1 to KCC2. Finally, we observed short-term depression of GABAA postsynaptic currents in immature neurons that was significantly and gradually abolished by P28. These findings reveal that in the developing BLA GABAergic transmission is highly dynamic, reaching maturity at the end of the first postnatal month.

Spike-timing precision and neuronal synchrony are enhanced by an interaction between synaptic inhibition and membrane oscillations in the amygdala.

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2-6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine.

A transcriptomic analysis of type I-III neurons in the bed nucleus of the stria terminalis.

The activity of neurons in the anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) plays a critical role in anxiety- and stress-related behaviors. Histochemical studies have suggested that multiple distinct neuronal phenotypes exist in the BNST(ALG). Consistent with this observation, the physiological properties of BNST(ALG) neurons are also heterogeneous, and three distinct cell types can be defined (Types I-III) based primarily on their expression of four key membrane currents, namely I(h), I(A), I(T), and I(K(IR)). Significantly, all four channels are multimeric proteins and can comprise of more than one pore-forming α subunit. Hence, differential expression of α subunits may further diversify the neuronal population. However, nothing is known about the relative expression of these ion channel α subunits in BNST(ALG) neurons. We have addressed this lacuna by combining whole-cell patch-clamp recording together with single-cell reverse transcriptase polymerase chain reaction (scRT-PCR) to assess the mRNA transcript expression for each of the subunits for the four key ion channels in Type I-III neurons of the BNST(ALG.) Here, cytosolic mRNA from single neurons was probed for the expression of transcripts for each of the α subunits of I(h) (HCN1-HCN4), I(T) (Ca(v)3.1-Ca(v)3.3), I(A) (K(v)1.4, K(v)3.4, K(v)4.1-K(v) 4.3) and I(K(IR)) (Kir2.1-Kir2.4). An unbiased hierarchical cluster analysis followed by discriminant function analysis revealed that a positive correlation exists between the physiological and genetic phenotype of BNST(ALG) neurons. Thus, the analysis segregated BNST(ALG) neurons into 3 distinct groups, based on their α subunit mRNA expression profile, which positively correlated with our existing electrophysiological classification (Types I-III). Furthermore, analysis of mRNA transcript expression in Type I-Type III neurons suggested that, whereas Type I and III neurons appear to represent genetically homologous cell populations, Type II neurons may be further subdivided into three genetically distinct subgroups. These data not only validate our original classification scheme, but further refine the classification at the molecular level, and thus identifies novel targets for potential disruption and/or pharmacotherapeutic intervention in stress-related anxiety-like behaviors.