Characterisation of wide field neurons in the superior colliculus using the Ntsr1-Cre GN209 mouse line

Abstract

Since Cajal established the neuron doctrine in the late nineteenth century, neuroscience has sought to classify the diversity of neurons and understand how they communicate. Advances in genetics, imaging, and electrophysiology have refined our ability to define neuronal types by integrating molecular identity, morphology, and physiological properties. In this thesis, we characterised genetically identified neuron types in the Ntsr1-Cre GN209 line and focused on wide-field neurons (WFNs) in the superior colliculus to examine their intrinsic features and communication mechanisms.

First, we mapped Cre-dependent expression throughout the brain and identified regions with strong labelling using Ntsr1-Cre GN 209 line. Whole-cell recordings and biocytin reconstructions showed that this line labels distinct neuronal classes, including Purkinje cells in the cerebellum, GABAergic interneurons in the inferior colliculus, WFNs in the superior colliculus, medium spiny neurons in the striatum, and pyramidal-like cells in the anterior olfactory nucleus, piriform cortex, and subiculum. Statistical analysis confirmed consistent within-region profiles and clear separation between classes, defining the cellular targets of the Ntsr1-Cre GN209 line and providing a validated reference for cell-specific research.

We next examined WFNs in detail. Most intrinsic properties, including resting membrane potential (RMP), input resistance, and action potential (AP) threshold, showed no developmental change after two weeks, while AP width, after-hyperpolarisation, and sag kinetics stabilised by six weeks, indicating early functional maturity. WFNs exhibited large voltage sags, depolarised RMP, and gradual RMP drift after depolarisation. Blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with ZD7288 reduced sag amplitude, hyperpolarised RMP, and suppressed RMP drift, showing that HCN activity dominates subthreshold dynamics. Morphological experiments using sparse viral labelling provided quantitative references for imaging precision and tissue swelling during clearing, laying groundwork for further structural analysis.

Finally, we investigated how WFNs communicate. Paired recordings showed that about 12% of pairs were electrically connected, including combinations of Cre positive and negative WFNs. Both depolarising and hyperpolarising responses were transmitted bidirectionally, indicating gap junctions between WFNs. Mefloquine reduced coupling without affecting intrinsic excitability, confirming a specific effect on gap junctions. Pharmacological and optogenetic tests showed that cadmium chloride blocked synaptic responses but not optogenetically evoked coupling, whereas mefloquine abolished electrical responses. Neurobiotin tracing revealed coupled cells beyond the Cre positive population, including neuronal and non-neuronal elements, suggesting that WFNs form a heterogeneous electrically connected network within the superficial colliculus. Translational profiling of Cre positive WFNs detected connexin 36 and connexin 43, indicating these connexins underlie the coupling. Functionally, coupled pairs fired synchronously with short delays, showing that gap junctions enable fast and coordinated activity among WFNs.

This work systematically maps and classifies Cre-labelled neurons across the brain using the Ntsr1-Cre GN209 line, providing a reference for future cell-type-specific studies and a quantitative framework for neuronal classification. It further defines the physiological and structural properties of WFNs and reveals that they form an electrically coupled network in the superficial superior colliculus. This discovery establishes a cellular basis for understanding how WFNs coordinate activity and offers a foundation for future studies exploring how such networks influence visually guided behaviours. Show more