Biology subjects

Packer, A. M.

Publications and source records attributed to Packer, A. M..

11 recordsLinked to original sources

bioRxiv2025

Desensitization of opsin responses during all-optical interrogation depends on imaging parameters

SignificanceThe combination of two-photon calcium imaging and two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipulating neural circuit activity in vivo. All-optical experiments often use red-shifted opsins in combination with green fluorescent reporters of neuronal activity. However, their excitation spectra still partially overlap, meaning that the imaging laser can excite the opsin. Though some care has been taken in the past to understand the effects of this spectral overlap, further work is required to understand its impact on the findings of all-optical studies. AimWe aimed to investigate whether two-photon imaging of the green fluorescent calcium reporter GCaMP6s at 920 nm increases the rate of desensitization in neurons expressing the red-shifted opsin C1V1. ApproachWe systematically varied either the inter-stimulus interval or the duration of two-photon calcium imaging during two-photon optogenetic stimulation of mouse layer 2/3 barrel cortex or visual cortex neurons. ResultsWe found that two-photon imaging at 920 nm increases the desensitization of photostimulation responses across trials in C1V1-expressing neurons - an effect that is exacerbated at shorter inter-stimulus intervals. Reduced photostimulation responses are not limited to targeted cells, and are found across the entire field of view. Such network effects are less pronounced at shorter imaging doses. ConclusionsOur results provide methodological optimizations that enable opsin desensitization to be mitigated in all-optical experiments. This will reduce an external source of trial-by-trial variability in future all-optical experiments.

neuroscience↗

bioRxiv2024

Perinatal serotonin signalling dynamically influences the development of cortical GABAergic circuits with consequences for lifelong sensory encoding

Serotonin plays a prominent role in neurodevelopment, regulating processes from cell division to synaptic connectivity1,2. Clinical studies suggest that alterations in serotonin signalling such as genetic polymorphisms3-5 or antidepressant exposure during pregnancy6,7 are risk factors for neurodevelopmental disorders. However, an understanding of how dysfunctional neuromodulation alters systems level activity over neocortical development is lacking. Here, we use a longitudinal imaging approach to investigate how genetics, pharmacology, and aversive experience disrupt state-dependent serotonin signalling with pathological consequences for sensory processing. We find that all three factors lead to increased neocortical serotonin levels during the initial postnatal period. Genetic deletion of the serotonin transporter or antidepressant dosing results in a switch from hypo- to hyper-cortical activity that arises as a consequence of altered cortical GABAergic microcircuitry. However, the trajectories of these manipulations differ with postnatal exposure to antidepressants having effects on adult sensory encoding. The latter is not seen in the genetic model despite a similar early phenotype, and a distinct influence of maternal genotype on the development of supragranular layers. These results reveal the dynamics and critical nature of serotonin signalling during perinatal life; pharmacological targeting of which can have profound life-long consequences for cognitive development of the offspring.

neuroscience↗

bioRxiv2024

The claustrum enhances neural variability by modulating the responsiveness of the prefrontal cortex

The claustrum is recognized for its significant impact on various cognitive functions and its extensive connections with other brain regions, yet its functional role remains to be fully understood. Here, we utilized an optogenetic approach to investigate the claustrums influence on neuronal activity within the dorsal prefrontal cortex (dPFC) of mice. We conducted two-photon calcium imaging to assess dPFC cell responses during exposure to visual stimuli and widefield photostimulation of claustrum axons embedded in the dPFC. We identified three distinct subpopulations of neurons -- sensory responsive, opto responsive, and opto-boosted cells -- each exhibiting unique response dynamics to combined visual and optogenetic stimuli. Our findings reveal that stimulation of claustrum axons can normalize neuronal responsiveness, while enhancing neural variability, and significantly increasing network homogeneity. Training in a Pavlovian task showed that while enhanced variability with claustrum axon stimulation in neural responses persists, training does not further increase this variability but instead leads to greater network homogeneity. Additionally, we also performed claustrum axon silencing experiments that revealed that the claustrum may operate bidirectionally to maintain enhanced variability and homogeneity in the prefrontal cortex. These results highlight the crucial role of the claustrum in dynamically modulating dPFC activity, impacting both neuronal variability and network synchronization.

neuroscience↗

bioRxiv2024

A latent pool of neurons silenced by sensory-evoked inhibition can be recruited to enhance perception

Which patterns of neural activity in sensory cortex are relevant for perceptual decision-making? To address this question, we used simultaneous two-photon calcium imaging and targeted two-photon optogenetics to probe barrel cortex activity during a perceptual discrimination task. Head-fixed mice discriminated bilateral whisker deflections and reported decisions by licking left or right. Two-photon calcium imaging revealed sparse coding of contralateral and ipsilateral whisker input in layer 2/3 while most neurons did not show task-related activity. Activating small groups of pyramidal neurons using two-photon holographic photostimulation evoked a perceptual bias that scaled with the number of neurons photostimulated. This effect was dominated by the optogenetic activation of a small number of non-coding neurons, which did not show sensory or motor-related activity during task performance. Patterned photostimulation also revealed potent recruitment of cortical inhibition during sensory processing, which strongly and preferentially suppressed non-coding neurons. Our results provide a novel perspective on the circuit basis for the sparse coding model of somatosensory processing in which a pool of non-coding neurons, selectively suppressed by strong network inhibition during whisker stimulation, can be recruited to enhance perception. HighlightsO_LIAll-optical interrogation of barrel cortex during bilateral whisker discrimination C_LIO_LISparse coding of contralateral and ipsilateral whisker information C_LIO_LISelective sensory-evoked inhibition helps ensure sparse coding C_LIO_LIOptogenetic recruitment of stimulus non-coding neurons can aid perception C_LI

neuroscience↗

bioRxiv2024

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

neuroscience↗

bioRxiv2023

Optical resolution is not the limiting factor for spatial precision of two-photon optogenetic photostimulation

Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable manipulation of neural circuits with a high degree of spatial precision. Combined with two-photon population calcium imaging, these approaches comprise a flexible platform for all-optical interrogation of neural circuits. However, a multitude of optical and biological factors dictate the exact precision of this approach in vivo, where it is most usefully applied. Here, we carefully assessed how the optical factors determine the spatial precision of activation. We found that optical resolution is not the limiting factor of the spatial precision of two-photon optogenetic photostimulation, and by doing so, reveal the key factors to improve to achieve maximal precision. Our results enable future work to focus on the optimal factors by providing key insight from controlled experiments in a manner not previously reported. This research can be applied to advance the state-of-the-art of all-optical interrogation, extending the toolkit for neuroscience research to achieve spatiotemporal precision at the crucial levels at which neural circuits operate.

neuroscience↗

bioRxiv2022

A multifaceted architectural framework of the mouse claustrum complex

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CC) this has yet to be fully accomplished. The CC is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are highly debated. To address this, we conducted a multifaceted analysis of fiber- and cyto-architecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to the CC. We identified four distinct subregions within the CC, subdividing both the CL and the DEn into two. Additionally, we conducted brain-wide tracing of inputs to the entire CC using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of the CC and its subregions. Myelinated fibers were abundant in dorsal parts of the CL but absent in ventral parts, while parvalbumin labelled fibers occupied the entire CL. Calbindin staining revealed a central gap within the CL, which was also visible at levels anterior to the striatum. Furthermore, cells in the CL projecting to the retrosplenial-cortex were located within the myelin sparse area. By combining our own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework. Significance statementMice are a highly tractable model for studying the claustrum complex (CC). However, without a consensus on how to delineate the CC in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CC, to match the level of detail needed to study its functional properties. Using multiple strategies for identifying claustral borders, we created a comprehensive guide to delineate the CC and its subregions. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CC.

neuroscience↗

bioRxiv2022

Temporal origin of mouse claustrum and development of its cortical projections

The claustrum is known for its extensive connectivity with many other forebrain regions, but its elongated shape and deep location have made further study difficult. We have sought to understand when mouse claustrum neurons are born, where they are located in developing brains and when they develop their widespread connections to cortex. We established that a well-characterised parvalbumin-plexus, which identifies the claustrum in adults, is only present from postnatal day (P)21. A myeloarchitectonic outline of the claustrum can be derived from a triangular fibre arrangement from P15. A dense patch of Nurr1+ cells is present at its core, and is already evident at birth. BrdU-birthdating of forebrain progenitors reveals that the majority of claustrum neurons are born during a narrow time window centred on embryonic day (E)12.5, which is later than the adjacent subplate and endopiriform nucleus. Retrograde tracing revealed that claustrum projections to anterior cingulate (ACA) and retrosplenial cortex (RSP) follow distinct developmental trajectories. Claustrum-ACA connectivity matures rapidly, and reaches adult-like innervation density by P10, whereas claustrum-RSP innervation emerges later over a protracted time window. This work establishes the timeline of claustrum development, and provides a framework for understanding how the claustrum is built and develops its unique connectivity.

neuroscience↗

bioRxiv2022

Single neurons and networks in the claustrum integrate input from widespread cortical sources

The claustrum is thought to be one of the most highly interconnected forebrain structures but its organizing principles have yet to be fully explored at the level of single neurons. Here, we investigated the identity, connectivity, and activity of identified claustrum neurons to understand how the structures unique convergence of input and divergence of output support binding information streams. We found that neurons in the claustrum communicate with each other across efferent projection-defined modules which were differentially innervated by sensory and frontal cortical areas. Individual claustrum neurons were responsive to inputs from more than one cortical region in a cell-type and projection-specific manner, particularly between areas of frontal cortex. In vivo imaging of claustrum axons revealed responses to both unimodal and multimodal sensory stimuli. Finally, chronic claustrum silencing specifically reduced animals sensitivity to multimodal stimuli. These findings support the view that the claustrum is a fundamentally integrative structure, consolidating information from around the cortex and redistributing it following local computations.

neuroscience↗

bioRxiv2021

Perception and propagation of activity through the cortical hierarchy is determined by neural variability

The brains of higher organisms are composed of anatomically and functionally distinct regions performing specialised tasks; but regions do not operate in isolation. Orchestration of complex behaviours requires communication between brain regions, but how neural activity dynamics are organised to facilitate reliable transmission is not well understood. We studied this process directly by generating neural activity that propagates between brain regions and drives behaviour, allowing us to assess how populations of neurons in sensory cortex cooperate to transmit information. We achieved this by imaging two hierarchically organised and densely interconnected regions, the primary and secondary somatosensory cortex (S1 and S2) in mice while performing two-photon photostimulation of S1 neurons and assigning behavioural salience to the photostimulation. We found that the probability of perception is determined not only by the strength of the photostimulation signal, but also by the variability of S1 neural activity. Therefore, maximising the signal-to-noise ratio of the stimulus representation in cortex relative to the noise or variability in cortex is critical to facilitate activity propagation and perception. Further, we show that propagated, behaviourally salient activity elicits balanced, persistent, and generalised activation of the downstream region. Hence, our work adds to existing understanding of cortical function by identifying how population activity is formatted to ensure robust transmission of information, allowing specialised brain regions to communicate and coordinate behaviour.

neuroscience↗

bioRxiv2019

The influence of visual cortex on perception is modulated by behavioural state

Our understanding of the link between neural activity and perception remains incomplete. Microstimulation and optogenetic experiments have shown that manipulating cortical activity can influence sensory-guided behaviour or elicit artificial percepts. And yet, some perceptual tasks can still be solved when sensory cortex is silenced or removed, suggesting that cortical activity may not always be essential. Reconciling these findings, and providing a quantitative framework linking cortical activity and behaviour, requires knowledge of the identity of the cells being activated during the behaviour, the engagement of the local and downstream networks, and the cortical and behavioural state. Here, we performed two-photon population calcium imaging in L2/3 primary visual cortex (V1) of headfixed mice performing a visual detection task while simultaneously activating specific groups of neurons using targeted two-photon optogenetics during low contrast visual stimulation. Only activation of groups of cells with similar tuning to the relevant visual stimulus led to a measurable bias of detection behaviour. Targeted photostimulation revealed signatures of centre-surround, predominantly inhibitory and like-to-like connectivity motifs in the local network which shaped the visual stimulus representation and partially explained the change in stimulus detectability. Moreover, the behavioural effects depended on overall performance: when the task was challenging for the mouse, V1 activity was more closely linked to performance, and cortical stimulation boosted perception. In contrast, when the task was easy, V1 activity was less informative about performance and cortical stimulation suppressed stimulus detection. Altogether, we find that both the selective routing of information through functionally specific circuits, and the prevailing cortical state, make similarly large contributions to explaining the behavioural response to photostimulation. Our results thus help to reconcile contradictory findings about the involvement of primary sensory cortex in behavioural tasks, suggesting that the influence of cortical activity on behaviour is dynamically reassigned depending on the demands of the task.

neuroscience↗