Biology subjects

Raimondo, J. V.

Publications and source records attributed to Raimondo, J. V..

16 recordsLinked to original sources

bioRxiv2025

Human neurons undergo protracted functional maturation into adulthood

Summary ParagraphHuman cognitive development is uniquely prolonged1-3, reflecting the extended postnatal maturation of the cerebral cortex where cell-type differentiation4,5, synaptogenesis6,7, myelination8 and transcriptional regulation5,9,10 all follow protracted developmental timelines. However, when human cortical neurons reach functional electrophysiological maturity and how their developmental trajectory compares to other species remains unknown. Here we show through patch-clamp recordings of human temporal cortex from infancy to adulthood that supragranular pyramidal neurons exhibit pronounced neoteny of their functional properties, with physiological maturation continuing well into adulthood. Comparing human and mouse developmental trajectories reveals human neurons are on a much slower developmental timeline, maturing physiologically hundreds of times slower than mouse and 2-6 times slower than would be predicted from anatomical brain growth differences between species. This reflects a fundamentally different allometric relationship between physiological and anatomical maturation; while mouse neuronal physiology closely tracks brain growth, human physiological development follows its own extended timeline. This slow maturation results in different stages of cognitive development being supported by functionally distinct neuronal populations, with the progression from infancy to middle age characterized by specific electrophysiological profiles. Notably, a neuronal subtype thought to be human-specific, with electrophysiological traits that enhance computational capacity, appears only in late adolescence or early adulthood. This extreme protraction of neurophysiological development provides a cellular basis for prolonged human cognitive maturation, demonstrating that neuronal physiological neoteny represents a fundamental evolutionary adaptation in human brain development.

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bioRxiv2024

Comprehensive Analysis of Human Dendritic Spine Morphology and Density: Exploring Diversity of Human Dendritic Spines

Dendritic spines, small protrusions on neuronal dendrites, play a crucial role in brain function by changing shape and size in response to neural activity. So far, in depth analysis of dendritic spines in human brain tissue is lacking. This study presents a comprehensive analysis of human dendritic spine morphology and density using a unique dataset from human brain tissue from 27 patients (8 females, 19 males, aged 18-71) undergoing tumor or epilepsy surgery at three neurosurgery sites. We used acute slices and organotypic brain slice cultures to examine dendritic spines, classifying them into the three main morphological subtypes: Mushroom, Thin, and Stubby, via 3D reconstruction using ZEISS arivis Pro software. A deep learning model, trained on 39 diverse datasets, automated spine segmentation and 3D reconstruction, achieving a 74% F1-score and reducing processing time by over 50%. We show significant differences in spine density by sex, dendrite type, and tissue condition. Females had higher spine densities than males, and apical dendrites were denser in spines than basal ones. Acute tissue showed higher spine densities compared to cultured human brain tissue. With time in culture, Mushroom spines decreased, while Stubby and Thin spine percentages increased, particularly from 7-9 to 14 days in vitro, reflecting potential synaptic plasticity changes. Our study underscores the importance of using human brain tissue to understand unique synaptic properties and shows that integrating deep learning with traditional methods enables efficient large-scale analysis, revealing key insights into sex- and tissue-specific dendritic spine dynamics relevant to neurological diseases. New and NoteworthyThis study presents a dataset of nearly 4,000 morphologically reconstructed human dendritic spines across different ages, gender, and tissue conditions. The dataset was further used to evaluate a deep learning algorithm for three-dimensional spine reconstruction, offering a scalable method for semi-automated spine analysis across various tissues and microscopy setups. The findings enhance understanding of human neurology, indicating potential connections between spine morphology, brain function, and the mechanisms of neurological and psychiatric diseases.

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bioRxiv2024

Network models incorporating chloride dynamics predict optimal strategies for terminating status epilepticus

Seizures that continue for beyond five minutes are classified as status epilepticus (SE) and constitute a medical emergency. Benzodiazepines, the current first-line treatment, attempt to terminate SE by increasing the conductance of chloride-permeable type-A GABA receptors (GABAARs). Despite their widespread use, benzodiazepines are ineffective in over a third of cases. Previous research in animal models has demonstrated that changes in intraneuronal chloride homeostasis and GABAAR physiology may underlie the development of benzodiazepine resistance in SE. However, there remains a need to understand the effect of these changes at a network level to improve translation into the clinical domain. Therefore, informed by data from human EEG recordings of SE and experimental brain slice recordings, we used a large spiking neural network model that incorporates chloride dynamics to investigate and address the phenomenon of benzodiazepine resistance in SE. We found that the GABAAR reversal potential (EGABA) sets SE-like bursting and determines the response to GABAAR conductance modulation, with benzodiazepines being anti-seizure at low EGABA and ineffective or pro-seizure at high EGABA. The SE-like activity and EGABA depended on a non-linear relationship between the strength of Cl- extrusion and GABAAR conductance, but not on the initial EGABA of neurons. Independently controlling Cl- extrusion in the pyramidal and interneuronal cell populations revealed the critical role of pyramidal cell Cl- extrusion in determining the severity of SE activity and the response to simulated benzodiazepine application. Finally, we demonstrate the models utility for considering improved therapeutic approaches for terminating SE in the clinic.

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bioRxiv2023

Cell type-specific gene expression dynamics during human brain maturation

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. Most studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-seq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 75 cell subtypes, which we verify with spatial transcriptomics. We explore the differences between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during its development and contribute to global efforts to build an inclusive brain cell map.

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bioRxiv2023

All-optical reporting of chloride ion driving force in the nervous system.

Ionic driving forces provide the net electromotive force for ion movement across receptors, channels, and transporters, and are a fundamental property of all cells. In the brain for example, fast synaptic inhibition is mediated by chloride permeable GABAA receptors, and single-cell intracellular recordings have been the only method for estimating driving forces across these receptors (DFGABAA). Here we present a new tool for quantifying inhibitory receptor driving force named ORCHID: all-Optical Reporting of CHloride Ion Driving force. We demonstrate ORCHIDs ability to provide accurate, high-throughput measurements of resting and dynamic DFGABAA from genetically targeted cell types over multiple timescales. ORCHID confirms theoretical predictions about the biophysical mechanisms that establish DFGABAA, reveals novel differences in DFGABAA between neurons and astrocytes, and affords the first in vivo measurements of intact DFGABAA. This work extends our understanding of inhibitory synaptic transmission and establishes a precedent for all-optical methods to assess ionic driving forces.

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bioRxiv2023

Active cortical networks promote shunting fast synaptic inhibition in vivo

Fast synaptic inhibition determines neuronal response properties in the mammalian brain and is mediated by chloride-permeable ionotropic GABA-A receptors (GABAARs). Despite their fundamental role, it is still not known how GABAARs signal in the intact brain. Here we use in vivo gramicidin recordings to investigate synaptic GABAAR signaling in mouse cortical pyramidal neurons under conditions that preserve native transmembrane chloride gradients. In anaesthetized cortex, synaptic GABAARs exert classic hyperpolarizing effects. In contrast, GABAAR-mediated synaptic signaling in awake cortex is found to be predominantly shunting. This is due to more depolarized GABAAR equilibrium potentials (EGABAAR), which are shown to result from the high levels of synaptic activity that characterize awake cortical networks. The EGABAAR observed in awake cortex can facilitate the decoupling of local networks, which improves the ability of the network to discriminate stimuli. Our findings therefore suggest that GABAAR signaling adapts to optimize cortical functions.

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bioRxiv2021

Chloride dynamics determine the optimal distribution of inhibitory synapses to minimise dendritic excitability

AO_SCPLOWBSTRACTC_SCPLOWMany neurons in the mammalian central nervous system have complex dendritic arborisations and active dendritic conductances that enable these cells to perform sophisticated computations. How dendritically targeted inhibition affects local dendritic excitability is not fully understood. Here we use computational models of branched dendrites to investigate where GABAergic synapses should be placed to minimise dendritic excitability over time. To do so, we formulate a metric we term the "Inhibitory Level" (IL), which quantifies the effectiveness of synaptic inhibition for reducing the depolarising effect of nearby excitatory input. GABAergic synaptic inhibition is dependent on the reversal potential for GABAA receptors (EGABA), which is primarily set by the transmembrane chloride ion (Cl-) concentration gradient. We, therefore, investigated how variable EGABA and dynamic chloride affects dendritic inhibition. We found that the inhibitory effectiveness of dendritic GABAergic synapses combines at an encircled branch junction, "inhibitory accumulation". The extent of inhibitory accumulation is dependent on the number of branches and location of synapses but is independent of EGABA. This inhibitory accumulation occurs even for very distally placed inhibitory synapses when they are hyperpolarising - but not when they are shunting. When accounting for Cl- fluxes and dynamics in Cl- concentration, we observed that Cl- loading is detrimental to inhibitory effectiveness. This enabled us to determine the most inhibitory distribution of GABAergic synapses which is close to - but not at - a shared branch junction. This distribution balances a trade-off between a stronger combined inhibitory influence when synapses closely encircle a branch junction with the deleterious effects of increased Cl- loading that occurs when inhibitory synapses are co-located.

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bioRxiv2020

Peripheral Taenia infection increases immunoglobulins in the central nervous system.

Human cysticercosis is a disease caused by larvae of the cestode Taenia solium. It is the most common cause of adult-acquired epilepsy world-wide where it exacts a debilitating toll on the health and well-being of affected communities. It is commonly assumed that the major symptoms associated with cysticercosis are a result of the direct presence of larvae in the brain. As a result, the possible effect of peripherally located larvae on the central nervous system are not well understood. To address this question, we utilised the Taenia crassiceps intra-peritoneal murine model of cysticercosis, where larvae are restricted to the peritoneal cavity. In this model, previous research has observed behavioural changes in rodents but not the development of seizures. Here we used ELISAs, immunoblotting and the Evans Blue test for blood-brain barrier permeability to explore the central effects of peripheral infection of mice with Taenia crassiceps. We identified high levels of parasite-targeting immunoglobulins in the serum of Taenia crassiceps infected mice. We show that the Taenia crassciceps larvae themselves also contain and release host immunoglobulins over time. Additionally, we describe, for the first time, significantly increased levels of IgG within the hippocampi of infected mice, which are accompanied by changes in blood-brain barrier permeability. However, these Taenia crassiceps induced changes were not accompanied by alterations to the levels of proinflammatory, pro-seizure cytokines in the hippocampus. These findings contribute to the understanding of systemic and neuroimmune responses in the Taenia crassiceps model of cysticercosis, with implications for the pathogenesis of human cysticercosis. Graphical abstract created with BioRender.com O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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bioRxiv2020

Taenia larvae possess distinct acetylcholinesterase profiles with implications for host cholinergic signalling

Larvae of the cestodes Taenia solium and Taenia crassiceps infect the central nervous system of humans. Taenia solium larvae in the brain cause neurocysticercosis, the leading cause of adult-acquired epilepsy worldwide. Relatively little is understood about how cestode-derived products modulate host neural and immune signalling. Acetylcholinesterases, a class of enzyme that degrade acetylcholine, are produced by a host of parasitic worms to aid their survival in the host. Acetylcholine is an important signalling molecule in both the human nervous and immune systems, with powerful modulatory effects on the excitability of cortical networks. Therefore, it is important to establish whether cestode derived acetylcholinesterases may alter host neuronal cholinergic signalling. Here we make use of multiple techniques to profile acetylcholinesterase activity in different extracts of both Taenia crassiceps and Taenia solium larvae. We find that the larvae of both species contain substantial acetylcholinesterase activity. However, acetylcholinesterase activity is lower in Taenia solium as compared to Taenia crassiceps larvae. Further, whilst we observed acetylcholinesterase activity in all fractions of Taenia crassiceps larvae, including on the membrane surface and in the excreted/secreted extracts, we could not identify acetylcholinesterases on the membrane surface or in the excreted/secreted extracts of Taenia solium larvae. Finally, using whole-cell patch clamp recordings in rat hippocampal brain slice cultures, we demonstrate that Taenia larval derived acetylcholinesterases can modify neuronal responses to acetylcholine. Together, these findings highlight the possibility that Taenia larval acetylcholinesterases can interfere with cholinergic signalling in the host, potentially contributing to pathogenesis in neurocysticercosis. Author summaryInfection of the human nervous system with larvae of the parasite Taenia solium is a significant cause of acquired epilepsy worldwide. Despite this, the precise cellular and molecular mechanisms underlying epileptogenesis in neurocysticercosis remain unclear. Acetylcholinesterases are a family of enzymes widely produced by helminthic parasites. These enzymes facilitate the breakdown of acetylcholine, which is also a major neurotransmitter in the human nervous system. If T. solium larvae produce acetylcholinesterases, this could potentially disrupt host cholinergic signalling, which may in turn contribute to seizures and epilepsy. We therefore set out to investigate the presence and activity of acetylcholinesterases in T. solium larvae, as well as in Taenia crassiceps larvae, a species commonly used as a model parasite in neurocysticercosis research. We found that both T. crassiceps and T. solium larvae produce acetylcholinesterases with substantial activity. We further demonstrate that the acetylcholinesterase activity in the products of these parasites is sufficient to disrupt cholinergic signalling in an ex vivo brain slice model. This study provides evidence that Taenia larvae produce acetylcholinesterases and that these can interfere with cholinergic signalling in the host and potentially contribute to pathogenesis in neurocysticercosis.

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bioRxiv2019

Cestode larvae excite host neuronal circuits via glutamatergic signaling.

Neurocysticercosis (NCC) is caused by infection of the brain by larvae of the parasitic cestode Taenia solium. It is the most prevalent parasitic infection of the central nervous system and one of the leading causes of adult-acquired epilepsy worldwide. However, little is known about how cestode larvae affect neurons directly. To address this, we used whole-cell patch-clamp electrophysiology and calcium imaging in rodent and human brain slices to identify direct effects of cestode larval products on neuronal activity. We found that both whole cyst homogenate and excretory/secretory products of cestode larvae have an acute excitatory effect on neurons, which can trigger seizure-like events in vitro. Underlying this effect was cestode-induced neuronal depolarization, which was mediated by glutamate receptor activation but not by nicotinic acetylcholine receptors, acid-sensing ion channels nor Substance P. Glutamate-sensing fluorescent reporters (iGluSnFR) and amino acid assays revealed that the larval homogenate of the cestodes Taenia crassiceps and Taenia solium contained high concentrations of the amino acids glutamate and aspartate. Furthermore, we found that larvae of both species consistently produce and release these excitatory amino acids into their immediate environment. Our findings suggest that perturbations in glutamatergic signaling may play a role in seizure generation in NCC.

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bioRxiv2019

Chloride dynamics alter the input-output properties of neurons

Fast synaptic inhibition is a critical determinant of neuronal output, with subcellular targeting of synaptic inhibition able to exert different transformations of the neuronal input-output function. At the receptor level, synaptic inhibition is primarily mediated by chloride-permeable Type A GABA receptors. Consequently, dynamics in the neuronal chloride concentration can alter the functional properties of inhibitory synapses. How differences in the spatial targeting of inhibitory synapses interact with intracellular chloride dynamics to modulate the input-output function of neurons is not well understood. To address this, we developed computational models of multi-compartment neurons that incorporate experimentally parametrised mechanisms to account for neuronal chloride influx, diffusion, and extrusion. We found that synaptic input (either excitatory, inhibitory, or both) can lead to subcellular variations in chloride concentration, despite a uniform distribution of chloride extrusion mechanisms. Accounting for chloride changes resulted in substantial alterations in the neuronal input-output function. This was particularly the case for peripherally targeted dendritic inhibition where dynamic chloride compromised the ability of inhibition to offset neuronal input-output curves. Our simulations revealed that progressive changes in chloride concentration mean that the neuronal input-output function is not static but varies significantly as a function of the duration of synaptic drive. Finally, we found that the observed effects of dynamic chloride on neuronal output were entirely mediated by changes in the dendritic reversal potential for GABA. Our findings provide a framework for understanding the computational effects of chloride dynamics on dendritically targeted synaptic inhibition.\n\nAuthor SummaryThe fundamental unit of computation in the brain is the neuron, whose output reflects information within the brain. A determining factor in the transfer and processing of information in the brain is the modulation of activity by inhibitory synaptic inputs. Fast synaptic inhibition is mediated by the neurotransmitter GABA binding to GABAA receptors, which are permeable to chloride ions. How changes in chloride ion concentration affect neuronal output is an important consideration for information flow in the brain that is currently not being thoroughly investigated. In this research, we used multi-compartmental models of neurons to link the deleterious effects that accumulation of chloride ions can have on inhibitory signalling with changes in neuronal ouput. Together, our results highlight the importance of accounting for changes in chloride concentration in theoretical and computer-based models that seek to explore the computational properties of inhibition.

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bioRxiv2019

Divergent paths to seizure-like events.

AimMuch debate exists about how the brain transitions into an epileptic seizure. One source of confusion is that there are likely to be critical differences between experimental seizure models. To address this, we compared the evolving activity patterns in two, widely used, in vitro models of epileptic discharges.\n\nMethodsWe compared brain slices, prepared in the same way from young adult mice, that were bathed either in 0 Mg2+, or 100{micro}M 4AP, artificial cerebrospinal fluid.\n\nResultsWe find that while local field potential recordings of epileptiform discharges in the two models appear broadly similar, patch-clamp analysis reveals an important difference in the relative degree of glutamatergic involvement. 4AP affects parvalbumin-expressing interneurons more than other cortical populations, destabilizing their resting state and inducing spontaneous bursting behavior. Consequently, the most prominent pattern of transient discharge (\"interictal event\") in this model is almost purely GABAergic, although the transition to seizure-like events (SLEs) involves pyramidal recruitment. In contrast, interictal discharges in 0 Mg2+ are only maintained by a very large glutamatergic component that also involves transient discharges of the interneurons. Seizure-like events in 0 Mg2+ have significantly higher power in the high gamma frequency band (60-120Hz) than these events do in 4AP, and are greatly delayed in onset by diazepam, unlike 4AP events.\n\nConclusionsThe 0 Mg2+ and 4AP models display fundamentally different levels of glutamatergic drive, demonstrating how ostensibly similar pathological discharges can arise from different sources. We contend that similar interpretative issues will also be relevant to clinical practice.

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bioRxiv2018

Excitatory GABAergic signalling is associated with acquired benzodiazepine resistance in status epilepticus

Status epilepticus (SE) is defined as a state of unrelenting seizure activity. It is associated with a rapidly rising mortality rate, and thus constitutes a medical emergency. Benzodiazepines, which act as positive modulators of chloride (Cl-) permeable GABAA receptors, are indicated as the first line of treatment, but this is ineffective in many cases. We found that 48% of children presenting with SE were unresponsive to benzodiazepine treatment, and critically, that the duration of SE at the time of treatment is an important predictor of non-responsiveness. We therefore investigated the cellular mechanisms that underlie acquired benzodiazepine resistance, using rodent organotypic brain slices. Removing Mg2+ ions leads to an evolving pattern of epileptiform activity, and eventually to a persistent state of repetitive discharges that strongly resembles clinical EEG recordings of SE. We show that the persistent SE-like activity is associated with a reduction in GABAA receptor conductance and Cl- extrusion capability. We explored the effect on intraneuronal Cl- using both gramicidin, perforated-patch clamp recordings and Cl- imaging. This showed that during SE-like activity, reduced Cl- extrusion capacity was further exacerbated by activity-dependent Cl- loading, resulting in a persistently high intraneuronal Cl-. Consistent with these results, we found that optogenetic stimulation of GABAergic interneurons in the SE-like state, actually enhanced epileptiform activity in a GABAAR dependent manner. Together our findings describe a novel potential mechanism underlying benzodiazepine-resistant SE, with relevance to how this life-threatening condition should be managed in the clinic.

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bioRxiv2018

Transcriptional and electrophysiological aberrations in an induced pluripotent stem cell-derived model of spinocerebellar ataxia type 7

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings demonstrate that iPSC-derived neurons and photoreceptors from SCA7 patients express molecular and electrophysiological differences that are indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.

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bioRxiv2017

Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis

Fast synaptic inhibition in the nervous system depends on the transmembrane flux of Cl- ions based on the neuronal Cl- driving force. Established theories regarding the determinants of Cl- driving force have recently been questioned. Here we present biophysical models of Cl- homeostasis using the pump-leak model. Using numerical and novel analytic solutions, we demonstrate that the Na+/K+-ATPase, ion conductances, impermeant anions, electrodiffusion, water fluxes and cation-chloride cotransporters (CCCs) play roles in setting the Cl- driving force. Our models, together with experimental validation, show that while impermeant anions can contribute to setting [Cl-]i in neurons, they have a negligible effect on the driving force for Cl- locally and cell-wide. In contrast, we demonstrate that CCCs are well-suited for modulating Cl- driving force and hence inhibitory signalling in neurons. Our findings reconcile recent experimental findings and provide a framework for understanding the interplay of different chloride regulatory processes in neurons.

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bioRxiv2016

Openspritzer: an open hardware pressure ejection system for reliably delivering picolitre volumes.

The ability to reliably and precisely deliver picolitre volumes is an important component of biological research. Here we describe a high-performance, low-cost, open hardware pressure ejection system (Openspritzer), which can be constructed from off the shelf components. When connected to a standard micro-pipette via suitable pneumatic tubing, the device is capable of delivering minute doses of reagents to a wide range of biological and chemical systems. In this work, we characterise the performance of the device and compare it to a popular commercial system using two-photon fluorescence microscopy. We found that Openspritzer provides the same level of control over delivered reagent dose as the commercial system. Next, we demonstrate the utility of Openspritzer in a series of standard neurobiological applications. First, we used Openspritzer to deliver precise amounts of the neurotransmitters glutamate and GABA to hippocampal neurons to elicit time- and dose-precise excitatory and inhibitory responses, respectively. Second, we used Openspritzer to deliver infectious viral and bacterial agents to living tissue. Viral transfection of hippocampal interneurons with channelrhodopsin allowed for the optogenetic manipulation of hippocampal circuitry with light. Finally, we successfully used Openspritzer to infect organotypic hippocampal slice cultures with fluorescent Mycobacterium bovis bacilli. We anticipate that due to its high performance and low cost Openspritzer will be of interest to a broad range of researchers working in the life and physical sciences.

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