CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

Journal of Neuroscience, 2018

Abstract:

The VTA is necessary for reward behavior with dopamine cells critically involved in reward signaling. Dopamine cells in turn are innervated and regulated by neighboring inhibitory GABA cells. Using whole-cell electrophysiology in juvenile-adolescent GAD67-GFP male mice, we examined excitatory plasticity in fluorescent VTA GABA cells. A novel CB1-dependent LTD was induced in GABA cells that was dependent on metabotropic glutamate receptor 5, and cannabinoid receptor 1 (CB1). LTD was absent in CB1 knock-out mice but preserved in heterozygous littermates. Bath applied Δ⁹-tetrahydrocannabinol depressed GABA cell activity, therefore downstream dopamine cells will be disinhibited; and thus, this could potentially result in increased reward. Chronic injections of Δ⁹-tetrahydrocannabinol occluded LTD compared with vehicle injections; however, a single exposure was insufficient to do so. As synaptic modifications by drugs of abuse are often tied to addiction, these data suggest a possible mechanism for the addictive effects of Δ⁹-tetrahydrocannabinol in juvenile-adolescents, by potentially altering reward behavioral outcomes.

Hippocampal Stratum Oriens Somatostatin-Positive Cells Undergo CB1-Dependent Long-Term Potentiation and Express Endocannabinoid Biosynthetic Enzymes.

Lindsey Friend, Williamson RC, Merrill CB, Newton ST, Christensen MT, Petersen J, Wu B, Ostlund I, Edwards JG.                                                                  Molecules, 2019

Abstract:

The hippocampus is thought to encode information by altering synaptic strength via synaptic plasticity. Some forms of synaptic plasticity are induced by lipid-based endocannabinoid signaling molecules that act on cannabinoid receptors (CB1). Endocannabinoids modulate synaptic plasticity of hippocampal pyramidal cells and stratum radiatum interneurons; however, the role of endocannabinoids in mediating synaptic plasticity of stratum oriens interneurons is unclear. These feedback inhibitory interneurons exhibit presynaptic long-term potentiation (LTP), but the exact mechanism is not entirely understood. We examined whether oriens interneurons produce endocannabinoids, and whether endocannabinoids are involved in presynaptic LTP. Using patch-clamp electrodes to extract single cells, we analyzed the expression of endocannabinoid biosynthetic enzyme mRNA by reverse transcription and then real-time PCR (RT-PCR). The cellular expression of calcium-binding proteins and neuropeptides were used to identify interneuron subtype. RT-PCR results demonstrate that stratum oriens interneurons express mRNA for both endocannabinoid biosynthetic enzymes and the type I metabotropic glutamate receptors (mGluRs), necessary for endocannabinoid production. Immunohistochemical staining further confirmed the presence of diacylglycerol lipase alpha, an endocannabinoid-synthesizing enzyme, in oriens interneurons. To test the role of endocannabinoids in synaptic plasticity, we performed whole-cell experiments using high-frequency stimulation to induce long-term potentiation in somatostatin-positive cells. This plasticity was blocked by AM-251, demonstrating CB1-dependence. In addition, in the presence of a fatty acid amide hydrolase inhibitor (URB597; 1 µM) and MAG lipase inhibitor (JZL184; 1 µM) that increase endogenous anandamide and 2-arachidonyl glycerol, respectively, excitatory current responses were potentiated. URB597-induced potentiation was blocked by CB1 antagonist AM-251 (2 µM). Collectively, this suggests somatostatin-positive oriens interneuron LTP is CB1-dependent.

Chronic Δ9-tetrahydrocannabinol impact on plasticity, and differential activation requirement for CB1-dependent long-term depression in ventral tegmental area GABA neurons in adult versus young mice.

Isaac Ostlund, Michael Von Gunten, Calvin Smith, Jeffrey G Edwards.                                                                                                                                            Frontiers in Neuroscience, 2023

Abstract:

The ventral tegmental area (VTA) mediates incentive salience and reward prediction error through dopamine (DA) neurons that are regulated by local VTA GABA neurons. In young mice, VTA GABA cells exhibit a form of synaptic plasticity known as long-term depression (LTD) that is dependent on cannabinoid 1 (CB1) receptors preceded by metabotropic glutamate receptor 5 (mGluR5) signaling to induce endocannabinoid production. This LTD was eliminated following chronic (7–10 consecutive days) exposure to the marijuana derived cannabinoid Δ9 -tetrahydrocannabinol (THC). We now examine the mechanism behind THC-induced elimination of LTD in adolescents as well as plasticity induction ability in adult versus young male and female mice using whole-cell electrophysiology experiments of VTA GABA cells. Chronic THC injections in adolescents resulted in a loss of CB1 agonist-mediated
depression, illustrating chronic THC likely desensitizes or removes synaptic CB1. We noted that seven days withdrawal from chronic THC restored LTD and CB1 agonist-induced depression, suggesting reversibility of THC-induced changes. Adult mice continue to express functional mGluR5 and CB1, but require a doubling of the synaptic stimulation compared to young mice to induce LTD, suggesting a quantitative difference in CB1-dependent plasticity between young and adult mice. One potential rationale for this difference is changes in AMPA and NMDA glutamate receptors. Indeed, AMPA/NMDA ratios were increased in in adults compared to young mice. Lastly, we performed quantitative reverse-transcription PCR and identified that CB1, DAGLα,
and GluA1 levels increased following chronic THC exposure. Collectively, our data demonstrate the first age-dependent GABA neuron plasticity in
the VTA, which could have implications for decreased THC dependence capacity in adults, as well as the mechanism behind chronic THC-induced
synaptic alterations in young mice.

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons.

Brittany A Davis, Huei-Ying Chen, Zengyou Ye, Isaac Ostlund, Madhavi Tippani, Debamitra Das, Srinidhi Rao Sripathy, Yanhong Wang, Jacqueline M
Martin, Gina Shim, Neel M Panchwagh, Rebecca L Moses, Federica Farinelli, Joseph F Bohlen, Meijie Li, Bryan W Luikart, Andrew E Jaffe, Brady J Maher.  Biological psychiatry, 2024

Abstract:

Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking.

Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons.

Automated high-throughput patch clamp electrophysiology of hiPSC-derived neuronal models.

Federica Farinelli, Isaac Ostlund, Srinidhi Rao Sripathy, Debamitra Das, Gina Shim, Sangho Myung, Richard E Straub, Brady J Maher.                                          bioRxiv 2025

Abstract:

The advent of human induced pluripotent stem cells (hiPSCs) and their differentiation into neurons and brain organoids has revolutionized our ability to model brain disorders in a human context. However, current technologies to assay the electrophysiological properties of human neurons in these models remain limited by throughput, as single-cell manual patch clamp is laborious and resource intensive. Here, we provide methods to perform high-throughput automated patch-clamp (APC) on hiPSC-derived neurons. We describe how to dissociate and perform voltage-clamp recordings on human neurons from three well-established protocols – 2D directed differentiation of cortical neurons, NGN2-induced neurons, and 3D cortical organoids – using the Nanion Syncropatch 384, a commercially available high-throughput APC system. Using this approach, we investigated the biophysical properties of voltage-gated sodium channels (VGSCs) and provide direct comparisons between manual and APC recordings across all three hiPSC-derived model systems. We demonstrate the capability of this automated system for pharmacological analysis of native human VGSC isoforms, which will enable compound screening approaches. Lastly, we provide methods to sort specific cellular populations within these hiPSC models using fluorescence-activated cell sorting (FACS) followed by APC. These methods and results provide a transformative and novel high-throughput technique for quantifying passive and active membrane properties in cell-type specific and/or genetically modified hiPSC-derived neurons.

OFFICE OF THE BRAIN DIRECTOR, NATIONAL INSTITUTES OF HEALTH Rockville, MD
Heath Program Specialist 12/01/2024 – 05/08/2025

Program Administrator
● Provided strategic budget planning and allocation guidance to stakeholders, including grant recipients and research laboratories, ensuring alignment with project scope, staffing requirements, and milestone deliverables.
● Navigated competing stakeholder demands and resolved conflicts to ensure proper task prioritization.
● Facilitated stakeholder engagement by coordinating workshops and project meetings involving subject matter
experts, researchers, government officials, and program officers; developed meeting agendas, briefing materials, and recorded action items to ensure alignment and accountability.
● Created public outreach activities to foster community awareness, BRAIN grant visibility, and stakeholder support.
● Performed comprehensive data analysis and strategic planning for neuroscience project portfolios, grant awards, and publication trends to generate executive-level reports and policy recommendations.
● Used Zoom, Teams, Outlook, Google calendar, Office, and more to schedule, communicate, and manage seminars, meetings, committees, and weekly reports.

LIEBER INSTITUTE FOR BRAIN DEVELOMENT, JOHNS HOPKINS UNIVERSITY BALTIMORE, MD
Staff Scientist 03/21/2022 – 12/01/2024

Lab Manager
● Created a multi-disciplinary team of cell biologists, electrophysiologists, and chemists to develop and perform research experiments examining neurodevelopmental disorders.
● Oversaw 2D and 3D IPSC projects, including growing organoids and managing cell cultures.
● Directed differentiation steps for IPSCs models and logistics from acquisition to storage and analysis, ensuring reproducibility and ethical compliance with lifecycle traceability and other requirements.
● Monitored the research progress of lab efforts to ensure scheduled milestones were being met and the final objective was attainable, reported these findings to leadership.
● Created and reviewed budgets to ensure sufficient resources to complete research and lab goals.
● Formed working teams and lasting professional relationships with internal groups and external biotechnological, government, and academic entities.
● Conducted meetings to discuss research needs, address concerns, and negotiate funding and resource allocation to overcome issues to reach project goals.
● Drafted data sharing agreements and material transfer agreements (MTAs), lab SOPs, and other key documents.
● Spearheaded workshops to teach IPSC requirements, methods, data QC, and lab SOP adherence.
● Led regular meetings to track grant progress and create draft versions of research performance progress reports (RPPRs).

Lab Researcher
● Conducted research using diverse experimental techniques including electrophysiology, histology, molecular biology, behavioral assays, and microscopy in both rodent and human neurons.
● Analyzed recent data and organized old data backlogs for accessibility and publication

BRIGHAM YOUNG UNIVERSITY PROVO, UT
Graduate Research Assistant 09/23/2016 – 04/16/2022

Undergraduate Supervisor and Project Manager
● Reviewed scientific literature and trends to determine novel venues of experimentation to create new research projects in plasticity, opioid mechanisms, neurocircuitry, and aging.
● Established research methodologies for fellow graduate and undergraduate lab members to follow to ensure data quality, control for variables, and produce reliable results.
● Managed and coordinated the efforts of 4 graduate and 26 undergraduate researchers.
● Pioneered the implementation of FAIR data principles in the lab, taught lab workshops to ensure full implementation of principles by lab cohort.
● Created lab research plans that assigned researchers their roles, set project goals, outlined needed resources, and then evaluated data generation for adequate progress.
● Served as a mediator to resolve conflicts and maintain research progress.
● Ensured schedule 1 drug regulatory adherence, animal care (IACUC), and other legal and policy requirement fulfillment by graduate and undergraduate researchers.
● Wrote reports to summarize the results and efforts of 26 undergraduate researchers to present to primary investigator and assess research outcomes.

Lab Researcher
● Designed and completed two major research projects discovering novel forms of age-dependent neuroplasticity and addiction mechanisms through slice electrophysiology, mouse stereotaxic surgery for viral injection, single cell RNA sequencing, and qRT-PCR.
● Analyzed how drugs of abuse, including opioids and marijuana, interplay with age to impact behavioral health, learning ability, and other public health issues.

SORENSON GENOMICS SALT LAKE CITY, UT
Lead Laboratory Technician 04/28/2014 – 03/17/2016

● Trained and managed a team of eight lab technicians, including monitoring their schedules, data reliability, and labproduction rates.
● Developed lab SOPs and maintained version histories, followed data QC tracking and requirements.
● Ensured SOP compliance and validation for all lab technicians to maintain quality assurance.
● Assisted Lab Director in managing orders, inventory, and other administrative tasks
● Performed high throughput DNA extraction, PCR, and analysis manually and through Biomek and Tecan
automated workstations.

PEER REVIEWED PUBLICATIONS AND CONFERENCE PRESENTATIONS
● Multiple first author papers in key journals, including the Journal of Neuroscience, Molecules, and Frontiers in Neuroscience.
● Multiple oral presentations at the Society for Neuroscience, Snowbird Neuroscience symposium, and others.

ADDITIONAL TRAINING
● 35 Hour Project Management Training. Certificate of Completion Received: July 2025. PMP in Progress.
● NIH Project Management Training Essentials Workshop, 2025
● NIH Technology Transfer & IP Essentials Workshop, 2024