Simon McMullan

Calcium-activated proteases, known as calpains, cleave proteins into smaller protein fragments, altering protein structure and function. Calpain overactivity is a pathological hallmark of many human diseases, including neurodegenerative diseases, which are characterized by excessive proteolytic cleavage and formation of protein aggregates. Current methods for determining calpain activity, such as examination of the presence or absence of calpain substrates in post-mortem tissue, are low-throughput, costly and retrospective, hampering the search for potentially therapeutically efficacious calpain inhibitor compounds. Here, we describe a novel methodology that allows real-time examination of calpain activity within the brain of the awake mouse. We used an adeno-associated viral (AAV) vector to express a fluorescence resonance energy transfer (FRET)-based calpain sensor in cerebellar neurons of male mice and measured real-time changes in FRET signal as a proxy for calpain activity via an implanted fibre optic cannula. This methodology was used to compare within-animal responses to different doses and administration routes of several calpain inhibitor compounds, including calpeptin, BLD-2736 and SNJ-1945. Using our AAV-calpain sensor and fibre photometry approach, we were able to obtain high-quality FRET recordings that were reproducible both between experimental animals and within experimental animals, allowing direct comparison of different calpain inhibitor compounds, enabling identification of the most efficacious treatment strategy that could be progressed further with preclinical treatment studies. As a positive control, we also tested changes in FRET signal in response to ionomycin, a known calcium ionophore. To our knowledge, this is the first report of real-time measurement of neuronal calpain activity in vivo. Future experiments could exploit this novel methodology to obtain calpain activity measurements alongside other pharmacological and behavioural/physiological measurements to increase the translational validity of preclinical models for drug development in diseases characterized by calpain overactivity.

The Allen Mouse Brain Atlas provides gene expression data for over 20,000 genes and has been extensively used in neuroscience. However, it’s constrained by a resolution of 200 µm³. We improved this to 25 µm³ for 4,083 genes by re-registering 401,660 sections to atlas space. From this, we produced an atlas with regions defined by shared molecular signatures and built an interactive online platform allowing exploration of gene expression patterns.

The Allen Mouse Brain Atlas provides gene expression data for over 20,000 genes and has been extensively used in neuroscience. However, it is constrained by a resolution of 200 µm ³ . We improved this to 25 µm ³ for 4,083 genes by re-registering 401,660 sections to atlas space. From this, we produced an atlas with regions defined by shared molecular signatures and built an interactive online platform allowing exploration of gene expression patterns.

Studies of the adult mouse brain have benefited from three-dimensional (3D) atlases providing standardised frameworks for data analysis and integration. However, few 3D atlases exist for the developing mouse brain, and none cover more than a few ages. This fails to represent the dynamic nature of development and is insufficient for researchers whose data fall between the ages of existent atlases. Existing atlases also neglect the spatial correspondence between ages, making comparison across ages difficult. We present the Developmental Mouse Brain Atlas (DeMBA), a 4D atlas encompassing every postnatal day from 4 to 56. DeMBA was created by using existing brain templates for six developmental time points and interpolating intermediate ages. DeMBA is incorporated in software for spatial registration and analysis, enabling brain-wide quantification of features of interest. Furthermore, we provide the software package CCF translator, allowing coordinates or image volumes to be transformed between ages for comparison. These resources provide age-specific spatial frameworks incorporated in accessible analytic pipelines for developmental mouse brain data.

The first phase of feeding consists in the procurement of solid foods from the environment by biting, and their preparation for swallowing by chewing. These actions require the precise coordination of tens of orofacial muscles for the jaw and tongue. The seat for this motor patterning is known to reside in the reticular formation, a complex and poorly mapped region of the hindbrain, but the neuron groups involved are still elusive. Here, we characterize a group of excitatory reticular interneurons located in the supratrigeminal area that express the homeodomain transcription factor Phox2b . This nucleus—Sup5 ^Phox2b —is premotor to both jaw-closing and jaw-opening motoneurons and receives direct input from cranial sensory afferents, motor cortex, and satiation related nuclei. Its activity differentially tracks lapping, biting, and chewing movements, suggesting its involvement in the elaboration of distinct orofacial motor patterns in vivo. Acute global activation or inhibition of Sup5 ^Phox2b by optogenetics interrupt volitional feeding sequences. Thus, Sup5 ^Phox2b is an obligatory subcortical node, topologically and genetically defined, in the neural circuits that control the oral phase of feeding in mice.

Oligodendrocyte progenitor cells (OPCs) receive synaptic input from a diverse range of neurons in the developing and adult brain. Understanding whether the neuronal populations that synapse with OPCs in the healthy brain is altered by demyelination and/or remyelination may support the advancement of neuroprotective or myelin repair strategies being developed for demyelinating diseases such as multiple sclerosis. To explore this possibility, we employed cre-lox transgenic technology to facilitate the infection of OPCs by a modified rabies virus, enabling the retrograde monosynaptic tracing of neuron→OPC connectivity. In the healthy adult mouse, OPCs in the corpus callosum primarily received synaptic input from ipsilateral cortical neurons. Of the cortical neurons, ∼50% were layer V pyramidal cells. Cuprizone demyelination reduced the total number of labeled neurons. However, the frequency/kinetics of mini-excitatory postsynaptic currents recorded from OPCs appeared preserved. Of particular interest, demyelination increased the number of labeled layer II/III pyramidal neurons and also increased at the expense of layer V pyramidal neurons, a change that was largely ameliorated by remyelination. These data suggest that in the healthy adult mouse brain, callosal OPCs primarily receive synaptic input from cortical layer V pyramidal neurons. However, callosal demyelination is associated with a population switch and OPCs equally synapse with layer II/III and V pyramidal neurons to synapse with OPCs, until myelin is restored.

Introduction: The subfornical organ (SFO) is a vital blood pressure-controlling region that elicits blood pressure changes likely via an excitatory (or glutamatergic) projection to the paraventricular nucleus of the hypothalamus (PVN). However, the role of this SFO-PVN pathway in blood pressure control has been poorly defined in the literature. As such, the present study aimed to examine the functional connectivity between the SFO neurons and the PVN and how they intersect to control blood pressure. Methods: In Lewis rats (n = 10), glutamatergic SFO neurons (SFOglut) were transduced with channelrhodopsin via a CaMKIIa-promotor vector (pAAV9-CaMKIIa-hChR2(H134R)-EYFP). Under urethane anaesthesia, changes in blood pressure and renal and splanchnic nerve activities were recorded in response to photostimulation of SFOglut neurons before and after administration of an intravenous ganglionic blocker and V1a receptor antagonism and inhibition of the PVN via muscimol microinjection. Immunohistochemistry was used to examine the projections between the SFO and PVN. Results: Photostimulation of SFOglut neurons produced a frequency-dependent pressor response that was abolished by sympathetic ganglionic blockade but not by inhibiting the vasoactive hormone vasopressin. This pressor response depends on ongoing neuronal transmission within the PVN as it is abolished by bilateral PVN inhibition. Confirming this, we found dense projections from SFOglut neurons to magnocellular and parvocellular PVN neurons. Finally, photostimulation of SFOglut neurons elicited a peak increase in sympathetic nerve activity that was reversibly abolished by phenylephrine administration and abolished by inhibition of the PVN, suggesting that the neuronal circuitry underpinning this response is barosensitive. Conclusion: The pressor response elicited by SFOglut neurons is largely mediated by barosensitive sympathetic nerve activity and dependent on the PVN.

Inhibition of neurons in the pontine Kölliker–Fuse/parabrachial complex (KF-PBN) differentially inhibited postinspiratory (post-I) activity in vagal and sympathetic outputs. The strong recruitment of post-I vagal activity that occurs in response to hypoxemia is selectively abolished by KF-PBN inhibition. This suggests that 1) post-I activity in vagal and sympathetic outputs may be generated by partially independent mechanisms and 2) neurons in the KF-PBN are a preeminent source of drive for the generation of eupneic post-I activity.

In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO₂)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO₂, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.

Registration of data to a common frame of reference is an essential step in the analysis and integration of diverse neuroscientific data. To this end, volumetric brain atlases enable histological datasets to be spatially registered and analyzed, yet accurate registration remains expertise-dependent and slow. In order to address this limitation, we have trained a neural network, DeepSlice, to register mouse brain histological images to the Allen Brain Common Coordinate Framework, retaining registration accuracy while improving speed by >1000 fold.