Biology subjects

Watson, J. F.

Publications and source records attributed to Watson, J. F..

9 recordsLinked to original sources

bioRxiv2026

Hippocampal CA3 forms a two-layer network of molecularly distinct cell types in mice and humans

Associational memory is critically dependent on the CA3 recurrent circuit, at the centre of the hippocampal system. While diversity amongst CA3 pyramidal neurons (PNs) has been long reported, the principles organising cell type identity and their consequences for circuit function remain unclear. Here, we demonstrate that deep-superficial is the primary axis of heterogeneity in CA3. PNs segregate into two molecularly distinct subtypes, superficial St18-, and deep St18+ neurons, with distinct anatomical, molecular, and functional properties. Using an St18-Cre mouse line, we reveal that these subtypes have divergent wiring patterns. Superficial neurons form the canonical broad autoassociative network across hemispheres and project to CA1, while deep St18+ PNs form a strikingly distinct, CA3-restricted, and predominantly unilateral projection -- a previously unrecognised circuit module embedded within the hippocampal associative network. This anatomical and molecular organisation is conserved across mammals, including mice, pigs, and humans, where patch-clamp recording in human tissue demonstrates conserved functional hallmarks of each PN subtype. Together, these findings fundamentally revise the hippocampal CA3 circuit map, revealing it as two highly distinct circuit elements and providing the molecular tools to dissect the contribution of each layer to hippocampal computation and cognition.

neuroscience↗

bioRxiv2024

'Mini analysis' is an unreliable reporter of synaptic changes

Analysis of miniature postsynaptic currents (mPSCs, or minis) is one of the most extensively employed approaches to determine the functional properties of synaptic connections. The popularity of this technique stems from its simplicity. Patch-clamp recording is used to observe spontaneous transmission occurring at synapses throughout a neurons dendritic tree. These events are analysed in an attempt to determine quantal synaptic parameters and changes during experimental manipulation. For decades, changes in the amplitude or frequency of mPSCs have been interpreted as evidence for specific synaptic modifications, including changes in presynaptic release sites, postsynaptic receptor abundance or even nanoscale changes in the alignment of synaptic machinery. However, at the majority of brain synapses, these events are small, with many undetectable due to recording noise. Here, using both simulated and experimental data, we demonstrate that interpreting synaptic changes from mPSC datasets is fundamentally fallible. Due to incomplete detection of event distributions, seemingly specific changes in mPSC amplitude or frequency falsely report actual synaptic changes. In addition, probabilistic detection of small events gives false confidence in the completeness of detection and inaccurate determination of quantal size. We not only demonstrate the dense pitfalls of mini analysis, but also establish a method for experimental detection of the detection limit, allowing more robust data analysis and scientific interpretation.

neuroscience↗

bioRxiv2024

Cell-specific wiring routes information flow through hippocampal CA3

The hippocampus is dogmatically described as a trisynaptic circuit. Dentate gyrus granule cells, CA3 pyramidal neurons (PNs), and CA1 PNs are serially connected, forming a circuit that critically enables memory storage in the brain. However, fundamental aspects of hippocampal function go beyond this simplistic trisynaptic definition. CA3 PNs connect not only to CA1, but also establish the largest autoassociative network in the brain. In addition, CA3 PNs are not uniform, differing in their morphology, intrinsic properties, and even the extent of granule cell input. Understanding how these different subtypes of CA3 PNs are embedded in the hippocampal network is essential for our quest to understand learning and memory. Here, we performed simultaneous multi-cellular patch-clamp recordings from up to eight CA3 PNs in acute mouse hippocampal slices, testing 3114 possible connections between identified cells. Combined with post-hoc morphological analysis, this allowed full characterization of neuronal heterogeneity in functioning microcircuits. We demonstrate that CA3 PNs can be divided into distinct deep and superficial subclasses, with altered input-output balance. While both subtypes formed recurrent connectivity within classes, connectivity between subtypes was surprisingly asymmetric. Recurrent connectivity was abundant from superficial to deep, but almost absent from deep to superficial PNs, thereby splitting CA3 into parallel recurrent networks which will allow more complex information processing. Finally, we observed innervation of PN subclasses by distinct interneurons, a potential mechanism to gate information flow through CA3 sublayers. Together, our data present a major revision to the classical trisynaptic view of the hippocampus, bringing us closer to understanding its complex action in information storage.

neuroscience↗

bioRxiv2024

Human hippocampal CA3 uses specific functional connectivity rules for efficient associative memory

The human brain has remarkable computational power. It generates sophisticated behavioral sequences, stores engrams over an individuals lifetime, and produces higher cognitive functions up to the level of consciousness. However, so little of our neuroscience knowledge covers the human brain, and it remains unknown whether this organ is truly unique, or is a scaled version of the extensively studied rodent brain. To address this fundamental question, we determined the cellular, synaptic, and connectivity rules of the hippocampal CA3 recurrent circuit using multicellular patch clamp-recording. This circuit is the largest autoassociative network in the brain, and plays a key role in memory and higher-order computations such as pattern separation and pattern completion. We demonstrate that human hippocampal CA3 employs sparse connectivity, in stark contrast to neocortical recurrent networks. Connectivity sparsifies from rodents to humans, providing a circuit architecture that maximizes associational power. Unitary synaptic events at human CA3-CA3 synapses showed both distinct species-specific and circuit-dependent properties, with high reliability, unique amplitude precision, and long integration times. We also identify differential scaling rules between hippocampal pathways from rodents to humans, with a moderate increase in the convergence of CA3 inputs per cell, but a marked increase in human mossy fiber innervation. Anatomically guided full-scale modeling suggests that the human brains sparse connectivity, expanded neuronal number, and reliable synaptic signaling combine to enhance the associative memory storage capacity of CA3. Together, our results reveal unique rules of connectivity and synaptic signaling in the human hippocampus, demonstrating the absolute necessity of human brain research and beginning to unravel the remarkable performance of our autoassociative memory circuits.

neuroscience↗

bioRxiv2022

Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics

AMPA glutamate receptors (AMPARs) mediate excitatory neurotransmission throughout the brain. Their signalling is uniquely diversified by brain region-specific auxiliary subunits, providing an opportunity for the development of selective therapeutics. AMPARs associated with TARP {gamma}8 are enriched in the hippocampus, and are targets of emerging anti-epileptic drugs. To understand their therapeutic activity, we determined cryo-EM structures of the GluA1/2-{gamma}8 receptor associated with three potent, chemically diverse drugs. We find that despite sharing a lipid-exposed and water-accessible binding pocket, drug action is differentially affected by binding-site mutants. Together with patch-clamp recordings and MD simulations we demonstrate that ligand-triggered reorganisation of the AMPAR-TARP interface contributes to modulation. Unexpectedly, one ligand (JNJ-61432059) acts bifunctionally, negatively affecting GluA1 but exerting positive modulatory action on GluA2-containing AMPARs, in a TARP stoichiometry-dependent manner. These results further illuminate the action of TARPs, demonstrate the sensitive balance between positive and negative modulatory action, and provide a mechanistic platform for development of both positive and negative selective AMPAR modulators.

neuroscience↗

bioRxiv2022

Uncovering brain tissue architecture across scales with super-resolution light microscopy

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the systems molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.

neuroscience↗

bioRxiv2022

Saturated reconstruction of living brain tissue

Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1-5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6-12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.

neuroscience↗

bioRxiv2020

Gating transitions and modulation of a hetero-octameric AMPA glutamate receptor

AMPA glutamate receptors (AMPARs) mediate the majority of excitatory transmission in the brain, and enable synaptic plasticity that underlies learning 1. A diverse array of AMPAR signaling complexes are established by receptor auxiliary subunits, associating in various combinations to modulate trafficking, gating and synaptic strength 2. However, their mechanisms of action are poorly understood. Here, we determine cryo-electron microscopy structures of the heteromeric GluA1/2 receptor assembled with both TARP-{gamma}8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states (at 3.2 and 3.7 [A], respectively). Consequential for gating regulation, two {gamma}8 and two CNIH2 subunits lodge at distinct sites beneath the ligand-binding domains of the receptor tetramer, with site-specific lipids shaping each interaction. Activation leads to a stark asymmetry between GluA1 and GluA2 along the ion conduction path, and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, that promotes the active-state conformation. In addition, both {gamma}8 and CNIH2 pivot towards the pore exit on activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this ER-export factor into a powerful positive AMPAR modulator, capable of providing hippocampal pyramidal neurons with their integrative synaptic properties.

neuroscience↗

bioRxiv2020

An inhalation anaesthesia approach for neonatal mice allowing streamlined stereotactic injection in the brain

Investigating brain function requires tools and techniques to visualise, modify and manipulate neuronal tissue. One powerful and popular method is intracerebral injection of customised viruses, allowing expression of exogenous transgenes. This technique is a standard procedure for adult mice, and is used by laboratories worldwide. Use of neonatal animals in scientific research allows investigation of developing tissues, and enables long-term study of cell populations. However, procedures on neonatal mice are more challenging, due to the lack of reliable methods and apparatus for anaesthesia of these animals. Here, we report an inhalation-based protocol for anaesthesia of neonatal (P0-2) mice, and present a custom 3D-printed apparatus for maintenance of anaesthesia during surgical procedures. This approach significantly enhances animal welfare and facilitates wider and simpler use of neonatal rodents in scientific research. Our optimised method of anaesthesia enables a rapid method of stereotactic injection in neonatal mice for transduction of brain tissue. We demonstrate this procedure for targeted labelling of specific brain regions, and in vivo modification of tissue prior to organotypic culture. This anaesthetic approach can be readily employed by any laboratory, and will enable safer use of neonatal rodents across a diverse spectrum of scientific disciplines. HighlightsO_LIDevelopment of inhalation-based anaesthesia for early postnatal (P0-2) mice C_LIO_LI3D-printed mould allows anaesthetic maintenance for neonatal surgery C_LIO_LIImproved mouse welfare through reliable neonatal inhalation anaesthesia C_LIO_LIRapid procedure for brain transduction of mouse litter in under 2 hours C_LI

neuroscience↗