Dustin J. Hines

Developmental epilepsy syndromes are characterized by recurrent seizures and developmental delays. Current anticonvulsants target γ-aminobutyric acid type A receptor signaling to decrease neuronal excitability, however, there are adverse effects for the developing brain, and many patients are refractory. The major non-psychotropic phytocannabinoid cannabidiol (CBD) has emerged as an anti-seizure medication effective in select developmental epilepsy syndromes, but its overall applicability in treating seizure disorders is limited. In the present study, we characterize a small library of non-Cannabis carvone derived CBD (+) enantiomers, with the larger goal of identifying novel therapeutics for developmental epilepsy syndromes. EEG based structure activity relationship assessment supports that elongated alkyl chains increase the potency of the congeners, with (+)-CBD-oct displaying effects on both δ and θ frequency bands. Pre-treatment with (+)-CBD-oct promotes seizure resilience in both wildtype mice and the Gabra2 -1 model of developmental epilepsy by influencing seizure characteristics, and reduces mortality. 5 days of (+)-CBD-oct oral gavage in wildtype and Gabra2 -1 mice during postnatal development normalizes the aberrant dendritic spine phenotype of Gabra2 -1 mice. These findings advance the development of novel anticonvulsants by validating an influence of alkyl chain length of synthetic CBD congeners.

Microglia are non-electrogenic immune cells that respond rapidly to protect the central nervous system (CNS) from infections, injuries, or other forms of damage. Microglia mitochondria are essential for providing the requisite energy resources for immune regulation. While fluctuations in energy metabolism are regulated by mitochondria and are reflected in the mitochondrial membrane potential (ΔΨm), there remains a lack of innovation in microglia-centric tools that capitalize on this. In this study, live imaging of microglia in acute slices from EGFP reporter mice expressing EGFP under the control of the fractalkine receptor (CX3CR1) promoter is combined with loading a fluorescent reporter of ΔΨm. Depolarizations in the ΔΨm were recorded after administering the well-characterized immune stimulant lipopolysaccharide (LPS). Microglia ΔΨm increased in distinctive phases with a relatively steep slope following LPS exposure. Conversely, the ΔΨm of neurons showed minimal regulation, highlighting a distinct microglia ΔΨm response to immune stimuli. Analysis of the depolarization of the microglia ΔΨm in the soma, branches, and endfeet revealed progressive changes in each subcellular domain originating in the soma and progressing outward. The inverse agonist emapunil attenuated the depolarization of the ΔΨm across states in a domain-specific manner. These findings emphasize the contribution of mitochondrial membrane dynamics in regulating microglial responses to immune stimuli. Further, this work advances a novel drug screening strategy for the therapeutic regulation of metabolic activity in inflammatory conditions of the brain.

A pulse generator using an avalanche transistor operating in current-mode second breakdown driving a 780 nm laser diode is reported. The laser diode is mounted on the skull of a mouse and used in transcranial infrared light stimulation (TILS) experiments. The output current pulse width is approximately 2 ns in an attempt to generate a true impulse-like optical pulse excitation for the TILS experiments.

Network communication in the CNS relies upon multiple neuronal and glial signaling pathways. In addition to synaptic transmission, other organelles such as mitochondria play roles in cellular signaling. One highly conserved mitochondrial signaling mechanism involves the 18 kDa translocator protein (TSPO) of the outer mitochondrial membrane. Originally, TSPO was identified as a binding site for benzodiazepines in the periphery. It was later discovered that TSPO is found in mitochondria, including in CNS cells. TSPO is implicated in multiple cellular processes, including the translocation of cholesterol and steroidogenesis, porphyrin transport, cellular responses to stress, inflammation, and tumor progression. Yet the impacts of modulating TSPO signaling on network activity and behavioral performance have not been characterized. In the present study, we assessed the effects of TSPO modulators PK11195, Ro5-4864, and XBD-173 via electroencephalography (EEG) and the open field test (OFT) at low to moderate doses. Cortical EEG recordings revealed increased power in the δ and θ frequency bands after administration of each of the three modulators, as well as compound- and dose-specific changes in α and γ. Behaviorally, these compounds reduced locomotor activity in the OFT in a dose-dependent manner, with XBD-173 having the subtlest behavioral effects while still strongly modulating the EEG. These findings indicate that TSPO modulators, despite their diversity, exert similar effects on the EEG while displaying a range of sedative/hypnotic effects at moderate to high doses. These findings bring us one step closer to understanding the functions of TSPO in the brain and as a target in CNS disease.

Intellectual disability (ID) is a common neurodevelopmental disorder that can arise from genetic mutations ranging from trisomy to single nucleotide polymorphism. Mutations in a growing number of single genes have been identified as causative in ID, including ARHGEF9. Evaluation of 41 ARHGEF9 patient reports shows ubiquitous inclusion of ID, along with other frequently reported symptoms of epilepsy, abnormal baseline EEG activity, behavioral symptoms, and sleep disturbances. ARHGEF9 codes for the Cdc42 Guanine Nucleotide Exchange Factor 9 collybistin (Cb), a known regulator of inhibitory synapse function via direct interaction with the adhesion molecule neuroligin-2 and the α2 subunit of GABA_A receptors. We mutate the Cb binding motif within the large intracellular loop of α2 replacing it with the binding motif for gephyrin from the α1 subunit (Gabra2-1). The Gabra2-1 mutation causes a strong downregulation of Cb expression, particularly at cholecystokinin basket cell inhibitory synapses. Gabra2-1 mice have deficits in working and recognition memory, as well as hyperactivity, anxiety, and reduced social preference, recapitulating the frequently reported features of ARHGEF9 patients. Gabra2-1 mice also have spontaneous seizures during postnatal development which can lead to mortality, and baseline abnormalities in low-frequency wavelengths of the EEG. EEG abnormalities are vigilance state-specific and manifest as sleep disturbance including increased time in wake and a loss of free-running rhythmicity in the absence of light as zeitgeber. Gabra2-1 mice phenocopy multiple features of human ARHGEF9 mutation, and reveal α2 subunit-containing GABA_A receptors as a druggable target for treatment of this complex ID syndrome.

Background: Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) play a critical role in neurodevelopment, where breast milk is a significant dietary source. The impact of previous COVID-19 infection and mastitis on the concentration of BDNF and NGF in human milk was investigated. Methods: Concentrations of BDNF and NGF were measured via ELISA in human milk samples collected from 12 mothers with a confirmed COVID-19 PCR, 13 mothers with viral symptoms suggestive of COVID-19, and 22 unexposed mothers (pre-pandemic Ctl-2018). These neurotrophins were also determined in 12 mothers with previous mastitis and 18 mothers without mastitis. Results: The NGF concentration in human milk was lower in the COVID-19 PCR and viral symptoms groups than in the unexposed group, but BDNF did not differ significantly. Within the COVID-19 group, BDNF was higher in mothers who reported headaches or loss of smell/taste when compared with mothers without the respective symptom. BDNF was lower in mothers with mastitis than in mothers without mastitis. Conclusions: Previous COVID-19 and mastitis infections changed differently the secretion of NGF and BDNF in human milk. Whether the changes in NGF and BDNF levels in milk from mothers with infection influence their infant’s development remains to be investigated.

Perception, emotion, and mood are powerfully modulated by serotonin receptor (5-HTR) agonists including hallucinogens. The 5-HT_2AR subtype has been shown to be central to hallucinogen action, yet the precise mechanisms mediating the response to 5-HT_2AR activation remain unclear. Hallucinogens induce the head twitch response (HTR) in rodents, which is the most commonly used behavioral readout of hallucinogen pharmacology. While the HTR provides a key behavioral signature, less is known about the meso level changes that are induced by 5-HT_2AR activation. In response to administration of the potent and highly selective 5-HT_2AR agonist 25I-NBOH in mice, we observe a disorganization of behavior which includes frequent episodes of behavioral arrest that consistently precede the HTR by a precise interval. By combining behavioral analysis with electroencephalogram (EEG) recordings we describe a characteristic pattern composed of two distinctive EEG waveforms, Phase 1 and Phase 2, that map onto behavioral arrest and the HTR respectively, with the same temporal separation. Phase 1, which underlies behavioral arrest, is a 3.5–4.5 Hz waveform, while Phase 2 is slower at 2.5–3.2 Hz. Nicotine pretreatment, considered an integral component of ritualistic hallucinogen practices, attenuates 25I-NBOH induced HTR and Phase 2 waveforms, yet increases behavioral arrest and Phase 1 waveforms. Our results suggest that in addition to the HTR, behavioral arrest and characteristic meso level slow waveforms are key hallmarks of the response to 5-HT_2AR activation. Increased understanding of the response to serotonergic hallucinogens may provide mechanistic insights into perception and hallucinations, as well as regulation of mood.

Route of administration is well-known to impact factors ranging from absorption and distribution, up through the subjective effects of active ingredients. Different routes of administration confer specific advantages, such as more rapid absorption resulting from intravenous injection, or increased convenience with oral administration, but a combination of both rapid and convenient delivery is highly desirable. QuickStrip™ was designed as a rapidly dissolving thin film matrix that contains active ingredients, which may be promising for rapid and convenient delivery via the oral mucosa. To assess the delivery of QuickStrip™, we administered the well-characterized active ingredient caffeine to mice and compared QuickStrip™ to standard oral gavage delivery at an equivalent dose of 20 mg kg^-1. Using HPLC assessment of serum concentrations of caffeine, we found that QuickStrip™ delivery resulted in higher serum levels of caffeine at 1, 10, and 30 min following administration compared to gavage. QuickStrip™ also produced greater bioavailability compared to gavage, as demonstrated by area under the curve analysis. Caffeine delivered by QuickStrip™ produced robust behavioral activation of locomotion, consistent with gavage caffeine. Electroencephalographic (EEG) assessment of central nervous system effects demonstrated that both gavage and QuickStrip™ caffeine produced suppression of delta and theta, consistent with prior literature on the effects of caffeine. In addition, QuickStrip™ produced a more rapid onset of EEG suppression, supporting the more rapid absorption demonstrated in the serum studies. Collectively, these studies suggest that QuickStrip™ may provide a balance between convenience and rapid onset, offering new options for delivery of therapeutics.

The brain is the most metabolically active organ in the body. This high metabolic demand is apparent in that 60% of the brain is comprised of mitochondria‐enriched cells. A disruption of the brain's ability to meet this immense metabolic demand is central to the pathogenesis of a multitude of neurological disorders, which range from depression to Alzheimer's disease. Central to these pathologies are glial signaling and energy metabolism cascades regulating apoptosis and inflammation. Thus, diseases causing inflammation and disruption of metabolism can be correlated with glial reactivity. Acutely, reactive gliosis provides a mechanism for limiting the progression of a disease. Following chronic activation, the ability of reactive gliosis to limit disease progression decreases and, in some cases, transitions into a harmful state. The necessity for a noninvasive biomarker of disease in the brain has linked reactive gliosis with an upregulation of translocator protein (TSPO). TSPO is an 18kDa protein that is both a therapeutic target for multiple acute and chronic neuroinflammatory diseases and the leading biomarker for Alzheimer's disease. Although a central function of TSPO is not well known, the protein was named for its ability to translocate cholesterol. Increased TSPO expression is an indicator of disrupted metabolic activity and increased reactive oxygen production. The changes in TSPO expression levels both temporally and spatially relate to the pathogenesis of stroke, Alzheimer's disease, traumatic brain injury, and depression. Therefore, research into the basic function and potential therapeutics targeting TSPO will have broad implications for many diseases of the brain.

Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizing γ2 subunit-containing GABA_Areceptors (GABA_ARs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABA_AR γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABA_AR γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.