Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4–8 simultaneously recorded neurons and/or 10–30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy–based, optogenetics- and imaging-assisted, stable, simultaneous quadruple–viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3–4 d.

Chandelier cells (ChCs; also called axo-axonic cells) are a specialized GABAergic interneuron subtype that selectively innervates pyramidal neurons at the axon initial segment (AIS), the site of action potential generation. ChC connectivity allows for powerful yet precise modulation of large populations of pyramidal cells, suggesting ChCs have a critical role in brain functions. Dysfunctions in ChC connectivity are associated with brain disorders such as epilepsy and schizophrenia; however, whether this is causative, contributory or compensatory is not known. A likely stumbling block toward mechanistic discoveries and uncovering potential therapeutic targets is the apparent lack of rudimentary understanding of ChCs. For example, whether cortical ChCs are inhibitory or excitatory remains unresolved, and thus whether altered ChC activity results in altered inhibition or excitation is not clear. Recent studies have shed some light onto this excitation-inhibition controversy. In addition, new findings have identified preferential cell-type connectivities established by cortical ChCs, greatly expanding our understanding of the role of ChCs in the cortical microcircuit. Here we aim to bring more attention to ChC connectivity to better understand its role in neural circuits, address whether ChCs are inhibitory or excitatory in light of recent findings and discuss ChC dysfunctions in brain disorders.

The hippocampus receives robust serotonergic innervation that is thought to control the excitability of both pyramidal cells and GABAergic interneurons. Previous work has addressed serotonergic regulation of pyramidal cells but considerable gaps remain in our understanding of how serotonin regulates different interneuron subclasses. 5-HT2A receptors (5-HT2ARs) appear to localize predominantly, if not solely, on interneurons in the hippocampus and have been implicated in the regulation of hippocampal function including mnemonic and novelty recognition processes. Interneurons are functionally diverse. Therefore in the current work, we have used a BAC transgenic mouse line expressing EGFP under the control of the 5-HT2AR promoter to identify the interneuron subtype(s) regulated by serotonin via 5-HT2ARs. We find that EGFP expression in this mouse identifies a group of interneurons that resides predominantly along the border of the stratum radiatum (SR) and stratum lacunosum moleculare (SLM) of the CA1 region. We then show that these cells are depolarized and excited by serotonin acting through 5-HT2ARs and appear to belong predominantly to the perforant pathway-associated and Schaffer collateral/commissural pathway-associated subtypes. These results indicate that serotonin interneurons expressing 5-HT2ARs are localized primarily along the SR-SLM border of the CA1 region and represent a newly identified target for serotonin regulation in the hippocampus.

Metabolism regulates neuronal activity and modulates the occurrence of epileptic seizures. Here, using two rodent models of absence epilepsy, we show that hypoglycemia increases the occurrence of spike-wave seizures. We then show that selectively disrupting glycolysis in the thalamus, a structure implicated in absence epilepsy, is sufficient to increase spike-wave seizures. We propose that activation of thalamic AMP-activated protein kinase (AMPK), a sensor of cellular energetic stress and potentiator of metabotropic GABAB receptor function, is a significant driver of hypoglycemia-induced spike-wave seizures. We show that AMPK augments postsynaptic GABAB receptor-mediated currents in thalamocortical neurons and strengthens epileptiform network activity evoked in thalamic brain slices. Selective thalamic AMPK activation also increases spike-wave seizures. Finally, systemic administration of metformin, an AMPK agonist and common diabetes treatment, profoundly increased spike-wave seizures. These results advance the decades-old observation that glucose metabolism regulates thalamocortical circuit excitability by demonstrating that AMPK and GABAB receptor cooperativity is sufficient to provoke spike-wave seizures.

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To understand how neuronal circuits underlie the processing of brain function, methods must be employed to dissect the circuit. Identification of the neuronal subtypes and characterization of how they interact are required to effectivity gain insight into understanding the neuronal circuit.The method to decipher neuronal circuits I describe and use here is the simultaneous multiple patch-clamp recording system. My overall aim was to elucidate interneuronal circuits in cortical layer 6 (L6), which are critical for specific brain processes such regulating sensory information.Studies on the connectivity of L6 have predominately focused on excitatory pyramidal neurons.Relatively little is known of the subtypes of GABAergic interneurons in L6 and how they are incorporated into the circuit. However, the role of L6 GABAergic inhibitory interneurons in brain function has recently become more appreciated.In this dissertation, I first provide background on L6 and basket cells, the most abundant GABAergic interneuron subtype. Then, in Chapters 2 and 3, I describe the simultaneous multiple patch-clamp recording system that is used to decipher interneuronal circuits. In Chapter 4, I examine a population of L6 interneurons using this method. Finally, I discuss my results and the field moving forward. ACKNOWLEDGMENTS