Calcium Imaging Recordings Research Articles

The neurokinin‐2 receptor antagonist GR 159897 protects against neuroinflammation in the mouse enteric nervous system during colitis

Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal (GI) disorder that affects 20% of Americans. The motility changes in IBS are driven by changes in the enteric nervous system (ENS). IBS has no cure, but recently, a neurokinin‐2 receptor (NK2R) antagonist has been shown to improve overall symptoms in a phase II study of diarrhea‐predominant IBS. However, the effects of NK2R antagonists on the ENS are not understood. We hypothesize that the beneficial effects of this antagonist on IBS are, in part, due to effects on the ENS. We tested our hypothesis by treating mice with the NK2R antagonist GR 159897 in a dinitrobenzene sulfonic acid (DNBS) model of colitis. To assess changes in the ENS, we used immunohistochemistry to quantify neuronal survival and performed a glial morphology analysis to assess reactive gliosis in the ENS. Calcium imaging recordings were performed in tissue preparations from mice expressing the genetically‐encoded calcium indicator GCaMP5g expressed under the control of the SOX10 promoter (SOX10::creERT2/+; PC::G5‐TdT+/−) or by loading cells with the calcium indicator dye Fluo4‐AM. Our results show that treatment with GR 159897 prevents increases in glial fibrillary acid protein (GFAP) immunoreactivity (n= 3–4 mice; p= 0.0141), increases in glial process length (n= 9–12 glia; p= 0.013) and neurodegeneration (n = 3–4; p= 0.0028). Immunohistochemical and calcium imaging data show that enteric glia express NK2Rs that are activated by neurokinin‐A (NKA). Glial responses to NKA were decreased in the presence of GR 159897 (n= 101–113 glia; p= 0.0001) or tetrodotoxin and were significantly reduced in samples from mice lacking glial connexin‐43 hemichannels (SOX10::creERT2/+; Cx43f/f mice; n= 50–113 glia; p= 0.0001). Glial responses to NKA were completely abolished in tissue samples from SOX10::creERT2/+; Cx43f/f mice in the presence of GR 159897 (n= 34–113; p= 0.0001). In conclusion, our data demonstrate that GR 159897 provides neuroprotection during colitis through mechanisms that involve decreasing glial activity driven by NKA. We speculate that these mechanisms could explain some of the clinical benefits of current NK2R antagonists in IBS.Support or Funding InformationRO1DK103723‐02S1

A Descending Neuron Correlated with the Rapid Steering Maneuvers of Flying Drosophila

To navigate through the world, animals must stabilize their path against disturbances and change direction to avoid obstacles and to search for resources [1, 2]. Locomotion is thus guided by sensory cues but also depends on intrinsic processes, such as motivation and physiological state. Flies, for example, turn with the direction of large-field rotatory motion, an optomotor reflex that is thought to help them fly straight [3-5]. Occasionally, however, they execute fast turns, called body saccades, either spontaneously or in response to patterns of visual motion such as expansion [6-8]. These turns can be measured in tethered flying Drosophila [3, 4, 9], which facilitates the study of underlying neural mechanisms. Whereas there is evidence for an efference copy input to visual interneurons during saccades [10], the circuits that control spontaneous and visually elicited saccades are not well known. Using two-photon calcium imaging and electrophysiological recordings in tethered flying Drosophila, we have identified a descending neuron whose activity is correlated with both spontaneous and visually elicited turns during tethered flight. The cell's activity in open- and closed-loop experiments suggests that it does not underlie slower compensatory responses to horizontal motion but rather controls rapid changes in flight path. The activity of this neuron can explain some of the behavioral variability observed in response to visual motion and appears sufficient for eliciting turns when artificially activated. This work provides an entry point into studying the circuits underlying the control of rapid steering maneuvers in the fly brain.

Immediate perception of a reward is distinct from the reward's long-term salience.

Reward perception guides all aspects of animal behavior. However, the relationship between the perceived value of a reward, the latent value of a reward, and the behavioral response remains unclear. Here we report that, given a choice between two sweet and chemically similar sugars-L- and D-arabinose-Drosophila melanogaster prefers D- over L- arabinose, but forms long-term memories of L-arabinose more reliably. Behavioral assays indicate that L-arabinose-generated memories require sugar receptor Gr43a, and calcium imaging and electrophysiological recordings indicate that L- and D-arabinose differentially activate Gr43a-expressing neurons. We posit that the immediate valence of a reward is not always predictive of the long-term reinforcement value of that reward, and that a subset of sugar-sensing neurons may generate distinct representations of similar sugars, allowing for rapid assessment of the salient features of various sugar rewards and generation of reward-specific behaviors. However, how sensory neurons communicate information about L-arabinose quality and concentration-features relevant for long-term memory-remains unknown.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network.

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons.

Somatostatin Interneurons Control a Key Component of Mismatch Negativity in Mouse Visual Cortex

Patients with schizophrenia have deficient sensory processing, undermining how they perceive and relate to a changing environment. This impairment can becaptured by the reduced mismatch negativity (MMN) index, an electroencephalographic biomarker of psychosis. The biological factors contributing to MMN are unclear, though mouse research, in which genetic and optical methods could be applied, has given some insight. Using fast two-photon calcium imaging and multielectrode recordings in awake mice, we find that visual cortical circuits display adapted (decreased) responses to repeated stimuli and amplified responses to a deviant stimulus, the key component of human MMN. Moreover, pharmacogenetic silencing of somatostatin-containing interneurons specifically eliminated this amplification, along with its associated theta/alpha-band response, leaving stimulus-specific adaption and related gamma-band modulations intact. Our results validate a mouse model of MMN and suggest that abnormalities in somatostatin-containing interneurons cause sensory deficits underlying MMN and schizophrenia.

Neuro-immune interactions in chemical-induced airway hyperreactivity.

Asthma may be induced by chemical sensitisers, via mechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivo model, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient Kit(Wsh) (/Wsh) mice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

Glucose Tightly Controls Morphological and Functional Properties of Astrocytes.

The main energy source powering the brain is glucose. Strong energy needs of our nervous system are fulfilled by conveying this essential metabolite through blood via an extensive vascular network. Glucose then reaches brain tissues by cell uptake, diffusion and metabolization, processes primarily undertaken by astrocytes. Deprivation of glucose can however occur in various circumstances. In particular, ageing is associated with cognitive disturbances that are partly attributable to metabolic deficiency leading to brain glycopenia. Despite the crucial role of glucose and its metabolites in sustaining neuronal activity, little is known about its moment-to-moment contribution to astroglial physiology. We thus here investigated the early structural and functional alterations induced in astrocytes by a transient metabolic challenge consisting in glucose deprivation. Electrophysiological recordings of hippocampal astroglial cells of the stratum radiatum in situ revealed that shortage of glucose specifically increases astrocyte membrane capacitance, whilst it has no impact on other passive membrane properties. Consistent with this change, morphometric analysis unraveled a prompt increase in astrocyte volume upon glucose deprivation. Furthermore, characteristic functional properties of astrocytes are also affected by transient glucose deficiency. We indeed found that glucoprivation decreases their gap junction-mediated coupling, while it progressively and reversibly increases their intracellular calcium levels during the slow depression of synaptic transmission occurring simultaneously, as assessed by dual electrophysiological and calcium imaging recordings. Together, these data indicate that astrocytes rapidly respond to metabolic dysfunctions, and are therefore central to the neuroglial dialog at play in brain adaptation to glycopenia.

Stimulus-induced pacemaker activity in interstitial cells of Cajal associated with the deep muscular plexus of the small intestine.

The ICC-DMP have been proposed to generate stimulus-dependent pacemaker activity, rhythmic transient depolarizations, that take part in orchestrating segmentation and clustered propulsive motor patterns in the small intestine. However, little is known about the fundamental properties of ICC-DMP. This study was undertaken to increase our understanding of intrinsic properties of the ICC-DMP through calcium imaging and intracellular electrical recordings. Without stimulation, most ICC-DMP were quiescent. In some preparations ICC-DMP generated rhythmic low-frequency calcium oscillations (<10 cpm) with or without high frequency activity superimposed (>35 cpm). Immunohistochemistry proved the existence of NK1R on the ICC-DMP and close contacts between ICC-DMP and substance P-positive nerves. Substance P (25 nM) induced low-frequency calcium oscillations that were synchronized across the ICC-DMP network. Substance P also induced low frequency rhythmic transient depolarizations (<10cpm) in circular muscle cells close to the ICC-DMP. An intracellular recording from a positively identified ICC-DMP showed rhythmic transient depolarizations with superimposed high frequency activity. To investigate if quiescent ICC-DMP were chronically inhibited by nitrergic activity, nNOS was inhibited, but without effect. Substance P changes non-synchronized high frequency flickering or quiescence in ICC-DMP into strong rhythmic calcium transients that are synchronized within the network; they are associated with rhythmic transient depolarizations within the same frequency range. We hypothesize that Substance P, released from nerves, can evoke rhythmicity in ICC-DMP, thereby providing it with potential pacemaker activity.

Electroactive polyurethane/siloxane derived from castor oil as a versatile cardiac patch, part II: HL-1 cytocompatibility and electrical characterizations.

In first part of this experiment, biocompatibility of the newly developed electroactive polyurethane/siloxane films containing aniline tetramer moieties was demonstrated with proliferation and differentiation of C2C12 myoblasts. Here we further assessed the cytocompatibility of the prepared samples with HL1-cell line, the electrophysiological properties and the patch clamp recording of the seeded cells over the selected electroactive sample. Presence of electroactive aniline tetramer in the structure of polyurethane/siloxane led to the increased expression of cardiac-specific genes of HL-1 cells involved in muscle contraction and electrical coupling. Our results showed that expression of Cx43, TrpT-2, and SERCA genes was significantly increased in conductive sample compared to tissue culture plate and the corresponding non-conductive analogous. The prepared materials were not only biocompatible in terms of cellular toxicity, but did not alter the intrinsic electrical characteristics of HL-1 cells. Embedding the electroactive moiety into the prepared films improved the properties of these polymeric cardiac construct through the enhanced transmission of electrical signals between the cells. Based on morphological observation, calcium imaging and electrophysiological recordings, we demonstrated the potential applicability of these materials for cardiac tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1398-1407, 2016.

Parallel Olfactory Processing in the Honey Bee Brain: Odor Learning and Generalization under Selective Lesion of a Projection Neuron Tract.

The function of parallel neural processing is a fundamental problem in Neuroscience, as it is found across sensory modalities and evolutionary lineages, from insects to humans. Recently, parallel processing has attracted increased attention in the olfactory domain, with the demonstration in both insects and mammals that different populations of second-order neurons encode and/or process odorant information differently. Among insects, Hymenoptera present a striking olfactory system with a clear neural dichotomy from the periphery to higher-order centers, based on two main tracts of second-order (projection) neurons: the medial and lateral antennal lobe tracts (m-ALT and l-ALT). To unravel the functional role of these two pathways, we combined specific lesions of the m-ALT tract with behavioral experiments, using the classical conditioning of the proboscis extension response (PER conditioning). Lesioned and intact bees had to learn to associate an odorant (1-nonanol) with sucrose. Then the bees were subjected to a generalization procedure with a range of odorants differing in terms of their carbon chain length or functional group. We show that m-ALT lesion strongly affects acquisition of an odor-sucrose association. However, lesioned bees that still learned the association showed a normal gradient of decreasing generalization responses to increasingly dissimilar odorants. Generalization responses could be predicted to some extent by in vivo calcium imaging recordings of l-ALT neurons. The m-ALT pathway therefore seems necessary for normal classical olfactory conditioning performance.

Simultaneous Fluorescent and Amperometric Detection Of Catecholamine Release From Neuroendocrine Cells With Transparent Diamond MEAs

Simultaneous Fluorescent and Amperometric Detection Of Catecholamine Release From Neuroendocrine Cells With Transparent Diamond MEAs

Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data

We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to invitro and invivo multi-neuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data.

Neural coding in barrel cortex during whisker-guided locomotion.

Animals seek out relevant information by moving through a dynamic world, but sensory systems are usually studied under highly constrained and passive conditions that may not probe important dimensions of the neural code. Here, we explored neural coding in the barrel cortex of head-fixed mice that tracked walls with their whiskers in tactile virtual reality. Optogenetic manipulations revealed that barrel cortex plays a role in wall-tracking. Closed-loop optogenetic control of layer 4 neurons can substitute for whisker-object contact to guide behavior resembling wall tracking. We measured neural activity using two-photon calcium imaging and extracellular recordings. Neurons were tuned to the distance between the animal snout and the contralateral wall, with monotonic, unimodal, and multimodal tuning curves. This rich representation of object location in the barrel cortex could not be predicted based on simple stimulus-response relationships involving individual whiskers and likely emerges within cortical circuits.

Involvement of aberrant calcium signalling in herpetic neuralgia

Alpha-herpesviruses, herpes simplex viruses (HSV) and varicella zoster virus (VZV), are pathogens of the peripheral nervous system. After primary infection, these viruses establish latency within sensory ganglia, while retaining the ability to reactivate. Reactivation of VZV results in herpes zoster, a condition characterized by skin lesions that leads to post-herpetic neuralgia. Recurrent reactivations of HSV, which cause mucocutaneous lesions, may also result in neuralgia. During reactivation of alpha-herpesviruses, satellite glial cells (SGCs), which surround neurons in sensory ganglia, become infected with the replicating virus. SGCs are known to contribute to neuropathic pain in a variety of animal pain models. Here we investigated how infection of short-term cultures of mouse trigeminal ganglia with HSV-1 affects communication between SGCs and neurons, and how this altered communication may increase neuronal excitability, thus contributing to herpetic neuralgia.Mechanical stimulation of single neurons or SGCs resulted in intercellular calcium waves, which were larger in cultures infected with HSV-1. Two differences were observed between control and HSV-1 infected cultures that could account for this augmentation. Firstly, HSV-1 infection induced cell fusion among SGCs and neurons, which would facilitate the spread of calcium signals over farther distances. Secondly, using calcium imaging and intracellular electrical recordings, we found that neurons in the HSV-1 infected cultures exhibited augmented influx of calcium upon depolarization.These virally induced changes may not only cause more neurons in the sensory ganglia to fire action potentials, but may also increase neurotransmitter release at the presynaptic terminals in the spinal cord. They are therefore likely to be contributing factors to herpetic neuralgia.

GABAergic inhibition shapes interictal dynamics in awake epileptic mice.

Epilepsy is characterized by recurrent seizures and brief, synchronous bursts called interictal spikes that are present in-between seizures and observed as transient events in EEG signals. While GABAergic transmission is known to play an important role in shaping healthy brain activity, the role of inhibition in these pathological epileptic dynamics remains unclear. Examining the microcircuits that participate in interictal spikes is thus an important first step towards addressing this issue, as the function of these transient synchronizations in either promoting or prohibiting seizures is currently under debate. To identify the microcircuits recruited in spontaneous interictal spikes in the absence of any proconvulsive drug or anaesthetic agent, we combine a chronic model of epilepsy with in vivo two-photon calcium imaging and multiunit extracellular recordings to map cellular recruitment within large populations of CA1 neurons in mice free to run on a self-paced treadmill. We show that GABAergic neurons, as opposed to their glutamatergic counterparts, are preferentially recruited during spontaneous interictal activity in the CA1 region of the epileptic mouse hippocampus. Although the specific cellular dynamics of interictal spikes are found to be highly variable, they are consistently associated with the activation of GABAergic neurons, resulting in a perisomatic inhibitory restraint that reduces neuronal spiking in the principal cell layer. Given the role of GABAergic neurons in shaping brain activity during normal cognitive function, their aberrant unbalanced recruitment during these transient events could have important downstream effects with clinical implications.

Evodiamine suppresses capsaicin-induced thermal hyperalgesia through activation and subsequent desensitization of the transient receptor potential V1 channels.

Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.

Preference for concentric orientations in the mouse superior colliculus.

The superior colliculus is a layered structure important for body- and gaze-orienting responses. Its superficial layer is, next to the lateral geniculate nucleus, the second major target of retinal ganglion axons and is retinotopically organized. Here we show that in the mouse there is also a precise organization of orientation preference. In columns perpendicular to the tectal surface, neurons respond to the same visual location and prefer gratings of the same orientation. Calcium imaging and extracellular recording revealed that the preferred grating varies with retinotopic location, and is oriented parallel to the concentric circle around the centre of vision through the receptive field. This implies that not all orientations are equally represented across the visual field. This makes the superior colliculus different from visual cortex and unsuitable for translation-invariant object recognition and suggests that visual stimuli might have different behavioural consequences depending on their retinotopic location.

TRPM3 expression in mouse retina.

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3-/- mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1‑/‑ mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3-/- mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

Neuroscience: The cortical connection.

Neurons in the brain's visual cortex receive inputs from thousands of other neurons. But it now emerges that each is strongly connected to only a few others: those most similar to itself. See Letter p.399 The degree to which a neuron influences the activity of others is dependent on the strength of the synaptic connections it makes with its partners, and it is known that this connection strength can vary over two orders of magnitude. Using a combination of two-photon calcium imaging and simultaneous intracellular recordings from pairs of neurons, Thomas Mrsic-Flogel and colleagues show that layer 2/3 neurons in mouse primary visual cortex (V1) follow a simple rule: strong connections are sparse and occur between neurons with correlated responses to visual stimuli, whereas only weak connections link neurons with uncorrelated responses. This bias in functional connection strength may be a means by which neuronal selectivity for visual features is computed in areas downstream of V1.

Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the cellular changes in the LDT induced by exposures to nicotine prenatally, when combined with alterations in other reward-related regions, could contribute to the increased susceptibility to smoking observed in the offspring.