Biology subjects

McDonald, A. J.

Publications and source records attributed to McDonald, A. J..

8 recordsLinked to original sources

bioRxiv2026

A highly Ca2+-permeable channelrhodopsin from the ancyromonad Nutomonas longa enables optogenetic control of Ca2+ signaling

Ca{superscript 2} is a ubiquitous regulator of cellular function, linking electrical activity to gene expression, secretion, metabolism, and synaptic plasticity. Yet, tools for its direct, time-resolved optical manipulation remain limited. Here, we report that NlCCR, a channelrhodopsin from Nutomonas longa, possesses high Ca{superscript 2} permeability, enabling precise optical control of Ca{superscript 2} signaling. Compared with CapChR2, the most potent engineered Ca{superscript 2}-conducting channelrhodopsin, NlCCR combines larger and faster photocurrents, higher Ca{superscript 2} permeability, weaker desensitization, and reduced inward rectification. Mutational analysis identified determinants of Ca{superscript 2} selectivity and further enhanced it by introducing carboxylate residues at the channels central gate. NlCCRs blue-shifted absorption (445 nm) minimized optical crosstalk with a red-shifted Ca{superscript 2} indicator, laying the groundwork for all-optical experiments. In mouse cortical pyramidal neurons, NlCCR enabled synaptic transmission independently of endogenous voltage-gated Ca{superscript 2} channels. These findings establish NlCCR as a broadly applicable tool for direct, temporally precise manipulation of Ca{superscript 2}-dependent signaling in living systems.

neuroscience↗

bioRxiv2026

A red-emitting, genetically encoded indicator for two-photon voltage recording in vivo

Genetically encoded voltage indicators (GEVIs) enable minimally invasive, cell-type-specific optical measurements of neuronal membrane potential with millisecond temporal resolution. Red-shifted GEVIs are especially advantageous because they permit spectral multiplexing with complementary sensors and enable all-optical circuit interrogation in combination with blue-light-activated opsins. Despite these advantages, existing red GEVIs remain poorly suited for in vivo use due to limited performance under two-photon (2P) excitation, the predominant modality for deep-tissue imaging. Here, we introduce VADER1, a red GEVI that overcomes this limitation and enables reliable spike detection in vivo under 2P illumination. Under 2P excitation, VADER1 supports extended voltage imaging with both random-access and resonant-scanning microscopy, enables recordings from neurons as deep as cortical layer 5, and allows dual-color imaging with calcium indicators. By filling a critical spectral gap, VADER1 enables integrated optical measurements of fast electrical activity alongside other neural signals and establishes a foundation for two-photon all-optical electrophysiology.

neuroscience↗

bioRxiv2026

Designer indicators for two-photon recording of subthreshold voltage dynamics

Subthreshold voltage dynamics are critical for neuronal information integration, yet they remain understudied in vivo due to the limitations of current tools. While genetically encoded voltage indicators (GEVIs) offer a promising alternative, their application for deep-tissue recording using two-photon (2P) microscopy--a preferred method for deep-tissue recording--has been hindered by insufficient sensitivity for detecting millivolt-scale subthreshold signals. Here, we refined our multiparametric two-photon high-throughput screening platform to develop two novel GEVIs, JEDI3sub and JEDI3hyp, tailored explicitly for subthreshold voltage detection. Through fast 2P optical recording in awake, behaving mice, we demonstrated the superior sensitivity of JEDI3 indicators compared to JEDI-2P. We also showed that JEDI3sub can track population-level subthreshold optical tuning, while JEDI3hyp reliably captured subthreshold dynamics associated with sharp-wave ripple oscillations in hippocampal PV interneurons. Finally, JEDI3hyp facilitated extended imaging of brain-state-dependent, millivolt-scale subthreshold voltage changes across deep-layer somas, fine dendritic structures, and diverse cell types. By addressing the critical gap in 2P optical recording of subthreshold voltage dynamics, JEDI3 indicators open new avenues for studying neural information processing and its alterations in health and disease.

neuroscience↗

bioRxiv2026

A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo

Genetically encoded voltage indicators (GEVIs) enable cell-type-specific optical readout of membrane potential, but two-photon (2P) spike detection has been hampered by low signal-to-noise and ultrafast off-kinetics, restricting use to specialized microscopes. We introduce FORCE1s, a green, positive-going GEVI engineered to make robust 2P voltage imaging broadly accessible. FORCE1s brightens from a dark baseline during depolarization, reports spikes with [~]100% {Delta}F/F in awake mice, and displays repolarization kinetics that are tuned for reliable spike detection at sub-kilohertz frame rates. As a result, FORCE1s supports spike-resolved multi-cell recordings on standard resonant-scanning microscopes, and further scales to larger fields of view and neuron counts on advanced modalities. FORCE1s also enables multiplexed voltage-neurotransmitter imaging and extended recordings in freely moving mice using a compact, affordable MEMS-based 2P miniscope. Together, these advances establish FORCE1s as a community-ready tool that democratizes deep-tissue voltage imaging across platforms and experimental contexts.

neuroscience↗

bioRxiv2025

Dual Role of LH-GABA Neurons in Encoding Alcohol Reward and Aversive Memories

One of the core aspects of alcohol use disorder is continued use despite negative consequences. Individuals with an alcohol use disorder typically engage in behaviors which represent a failure to integrate the aversive consequences of their actions. The neural mechanisms of learning about conflicting experiences remain poorly understood. Previous research has highlighted the critical role of GABAergic neurons in the Lateral Hypothalamus (LH-GABA) in rewarding memories. However, whether this role extends to aversive outcomes, and competition between rewarding and aversive outcomes remains unexplored. In this study, we sought to elucidate the role of LH-GABA neurons in encoding and expressing alcohol reward and aversive memories using fiber photometry calcium imaging. We used a dual-virus approach to confine expression of jGCaMP7f to GABAergic neurons in LH of male and female Long-Evans rats. In our first experiment, following acquisition of a cue-alcohol association, a mild foot shock was introduced on 50% of the trials. In the second experiment, we used three different cues to signal alcohol, foot-shock, or no consequence. Subsequently, we combined these conditioned stimuli in pairs to evoke motivational conflict. Our results reveal that LH-GABA activity is associated with cues predictive of both appetitive and aversive outcomes and is highest to a cue predictive of an aversive outcome. Additionally, the response of LH-GABA activity to an aversive shock stimulus is attenuated in the presence of alcohol. In summary, our findings show the activity of LH-GABA neurons is involved in learning both appetitive and aversive associations, and their interaction in conflict. Significance statement.This study addresses a critical knowledge gap in understanding the neural mechanisms behind learning in conflicting situations. Focusing on the GABAergic neurons in Lateral Hypothalamus, which have previously been associated with rewarding memories and motivation, we explore their role in the competition between rewarding and aversive motivation. We used fiber photometry to record LH-GABA neurons and found that their activity is heightened for aversive outcomes, irrespective of certainty. Notably, in situations of motivational conflict, this activity to the shock is dampened in the presence of alcohol. These insights not only enhance our understanding of the neural basis of emotional adaptation but also hold implications for psychiatric disorders like alcohol use disorder, depression, and anxiety, providing potential avenues for therapeutic interventions.

neuroscience↗

bioRxiv2023

Lateral hypothalamic GABAergic neurons encode alcohol memories.

In alcohol use disorder the alcohol memories persist during abstinence, and exposure to stimuli associated with alcohol use can lead to relapse. This highlights the importance of investigating the neural substrates underlying not only relapse, but also encoding and expression of alcohol memories. GABAergic neurons in the Lateral Hypothalamus (LH- GABA) have been shown to be critical for food-cue memories and motivation, however the extent to which this role extends to alcohol-cue memories and motivations remains unexplored. In this study, we aimed to describe how alcohol related memories are encoded and expressed in LH GABAergic neurons. Our first step was to monitor LH-GABA calcium transients during acquisition, extinction, and reinstatement of an alcohol-cue memory using fiber photometry. We trained the rats on a Pavlovian conditioning task where one conditioned stimulus (CS+) predicted alcohol (20% EtOH) and another conditioned stimulus (CS-) had no outcome. We then extinguished this association through non-reinforced presentations of the CS+ and CS-, finally in two different groups we measured relapse under non-primed and alcohol primed induced reinstatement. Our results show that initially both cues caused increased LH-GABA activity, and after learning only the alcohol-cue increased LH-GABA activity. After extinction this activity decreases, and we found no differences in LH- GABA activity during reinstatement in either group. Next, inhibited LH-GABA neurons with optogenetics to show that activity of these neurons is necessary for the formation of an alcohol-cue associations. These findings suggest that LH-GABA might be involved in attentional processes modulated by learning.

neuroscience↗

bioRxiv2023

Acetylcholine engages distinct amygdala microcircuits to gate internal theta rhythm

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in basolateral amygdala (BLA) underlie emotional memory, yet their mechanism remains unclear. Using brain slice electrophysiology in mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potential that depend upon activation of cholecystokinin (CCK) interneurons (INs). Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, gating BLA theta. ACh-induced theta activity is more readily evoked in BLA over cortex or hippocampus, suggesting preferential activation of BLA during high ACh states. These data reveal a SOM-CCK IN circuit in BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

neuroscience↗

bioRxiv2021

Differential regulation of prelimbic and thalamic transmission to the basolateral amygdala by acetylcholine receptors

The amygdalar anterior basolateral nucleus (BLa) plays a vital role in emotional behaviors. This region receives dense cholinergic projections from basal forebrain which are critical in regulating neuronal activity and synaptic transmission. Cholinergic signaling in BLa is thought to occur through both a slow mode of volume transmission as well as a rapid, phasic mode. However, the relative effect of each mode of signaling in BLa is not understood. Here, we used electrophysiology and optogenetics in mouse brain slices to compare regulation of afferent input from cortex and thalamus to the BLa by these two modes of transmission. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on glutamatergic transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower muscarinic receptor (mAChR)-mediated depression. In contrast, tonic elevation of ACh through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission at cortical inputs through mAChRs only. This suppression was not observed at thalamic inputs to BLa. In agreement with this pathway-specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex (PL) than thalamus. Muscarinic inhibition at PL input was dependent on presynaptic M4 mAChRs, while at thalamic input it depended upon M3 mAChR-mediated stimulation of retrograde endocannabinoid signaling. Muscarinic inhibition at both pathways was frequency-dependent, allowing only high frequency activity to pass. These findings demonstrate complex cholinergic regulation of afferent input to BLa that depends upon the mode of ACh release and is both pathway specific and frequency dependent. Significance statementCholinergic modulation of the basolateral amygdala regulates formation of emotional memories, but the mechanisms underlying this regulation are not well understood. Here, we show, using mouse brain slices, that ACh differentially regulates afferent transmission to the BLa depending on the mode of cholinergic signaling. Rapid, phasic ACh produces a biphasic excitatory-inhibitory regulation of glutamatergic transmission mediated by nicotinic and muscarinic receptors, respectively. In contrast, slow, tonic ACh produces muscarinic inhibition only. Tonic regulation is pathway specific with cortical input regulated more strongly than thalamic input. This disparity is caused by differential regulation by M4 and M3 receptors at the two inputs. Specific targeting of these receptors may thus provide a therapeutic strategy to bias amygdalar processing and regulate emotional memory.

neuroscience↗