Single Image-Based Vignetting Correction for Improving the Consistency of Neural Activity Analysis in 2-Photon Functional Microscopy

High-resolution functional 2-photon microscopy of neural activity is a cornerstone technique in current neuroscience, enabling, for instance, the image-based analysis of relations of the organization of local neuron populations and their temporal neural activity patterns. Interpreting local image intensity as a direct quantitative measure of neural activity presumes, however, a consistent within- and across-image relationship between the image intensity and neural activity, which may be subject to interference by illumination artifacts. In particular, the so-called vignetting artifact—the decrease of image intensity toward the edges of an image—is, at the moment, widely neglected in the context of functional microscopy analyses of neural activity, but potentially introduces a substantial center-periphery bias of derived functional measures. In the present report, we propose a straightforward protocol for single image-based vignetting correction. Using immediate-early gene-based 2-photon microscopic neural image data of the mouse brain, we show the necessity of correcting both image brightness and contrast to improve within- and across-image intensity consistency and demonstrate the plausibility of the resulting functional data.

Exercise Increases NPY/AgRP and TH Neuron Activity in the Hypothalamus of Female Mice

Recent evidence identifies a potent role for aerobic exercise to modulate activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low (10m/min), moderate (14m/min), and high (18m/min) intensity treadmill exercise in a randomized crossover design. Mice were fasted 10-hours before exercise and food intake was monitored for 48-hours after bouts. Immunohistochemical detection of cFOS was performed 3-hours post-exercise to determine changes in hypothalamic NPY/AgRP, POMC, tyrosine hydroxylase, and SIM1-expressing neuron activity concurrent with changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate and high intensity exercise increased 24-hour food intake by 5.9% and 19%, respectively, while low intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and tyrosine hydroxylase neuron activity were observed 3-hours after high intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3-hours post-exercise. Overall, aerobic exercise increased activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.

Transcriptional adaptation of sensory neurons to GPCR identity and activity

Sensory adaptation is critical to extract information from a changing world. Taking advantage of the extensive parallel coding lines present in the olfactory system, we explored the potential variations of neuronal identities before and after olfactory experience. We found that at rest, the transcriptomic profiles of olfactory sensory neuron populations are already highly divergent, specific to the olfactory receptor they express, and are surprisingly associated with the sequence of these latter. These divergent profiles further evolve in response to the environment, as odorant exposure leads to massive reprogramming via the modulation of transcription. Adenylyl cyclase 3, but not other main elements of the olfactory transduction cascade, plays a critical role in this activity-induced transcriptional adaptation. These findings highlight a broad range of sensory neuron identities that are present at rest and that adapt to the experience of the individual, thus providing a novel layer of complexity to sensory coding.

Odorant receptor copy number change, co-expression, and positive selection establish peripheral coding differences between fly species

Animals sample their chemical environment using sensory neurons that express diverse chemosensory receptors, which trigger responses when they bind environmental molecules. In addition to modifications in the ligand binding properties of receptors, chemosensory receptor evolution is characterized by copy number changes, often resulting in large gene family size differences between species. Though chemosensory receptor expansions and contractions are frequently described, it is unknown how this is accompanied by changes in the neural circuitry in which they are expressed. Among Drosophila's chemosensory receptor families, the Odorant receptors (Ors) are ideal for addressing this question because, other than an essential co-receptor (Orco), a large majority of Ors are uniquely expressed in single olfactory sensory neuron (OSN) populations. Between-species changes in Or copy number, therefore, may indicate diversification or reduction of peripheral sensory neuron populations. To test this possibility, we focused on a rapidly duplicated/deleted Or subfamily - named Or67a - within Drosophila melanogaster and its most closely-related sister species (D. simulans, D. sechellia, and D. mauritiana). Evolutionary genetic analyses and in vivo physiological assays demonstrate that the common ancestor of these four species possessed three Or67a paralogs that had already diverged adaptively in their odor-evoked responses. Following the group's speciation events, two Or67a paralogs were independently lost in D. melanogaster and D. sechellia, with positive selection continuing to act on the intact genes. Instead of the expected singular expression of each of the functionally diverged Ors in different neurons, we found that the three D. simulans Or67a paralogs are co-expressed in the same cells. Thus, while neuroanatomy is conserved between these species, independent selection on co-expressed receptors has contributed to species-specific peripheral coding of olfactory information. This work reveals a model of adaptive change previously not considered for olfactory evolution and raises the possibility that similar processes may be operating among the largely uninvestigated cases of Or co-expression.

Synaptic and intrinsic mechanisms impair reticular thalamus and thalamocortical neuron function in a Dravet syndrome mouse model

ABSTRACTThalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced intrinsic excitability in nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced ascending glutamatergic sensory input to VPL neurons, but not VPM neurons. In addition, glutamatergic cortical input to nRT neurons was reduced in Scn1a heterozygous mice, whereas cortical input to VPL and VPM neurons remained unchanged. These findings introduce input-specific alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in Dravet syndrome, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in Dravet syndrome and identifies new components of corticothalamic circuitry as potential therapeutic targets.HIGHLIGHTSGABAergic reticular thalamus neurons have impaired tonic and burst firing properties in a NaV1.1 haploinsufficiency mouse model of Dravet syndrome.NaV1.1 haploinsufficiency has opposing effects on spike firing in two distinct glutamatergic thalamocortical neuron populations.NaV1.1 haploinsufficiency alters glutamatergic synaptic connectivity in an input-specific manner in the thalamus.Dysregulation of both intrinsic and synaptic mechanisms contribute to imbalanced thalamic excitation and inhibition in this Dravet syndrome mouse model.

Spatial and Temporal Organization of Composite Receptive Fields in the Songbird Auditory Forebrain

The mechanisms underlying how single auditory neurons and neuron populations encode natural and acoustically complex vocal signals, such as human speech or bird songs, are not well understood. Classical models focus on individual neurons, whose spike rates vary systematically as a function of change in a small number of simple acoustic dimensions. However, neurons in the caudal medial nidopallium (NCM), an auditory forebrain region in songbirds that is analogous to the secondary auditory cortex in mammals, have composite receptive fields (CRFs) that comprise multiple acoustic features tied to both increases and decreases in firing rates. Here, we investigated the anatomical organization and temporal activation patterns of auditory CRFs in European starlings exposed to natural vocal communication signals (songs). We recorded extracellular electrophysiological responses to various bird songs at auditory NCM sites, including both single and multiple neurons, and we then applied a quadratic model to extract large sets of CRF features that were tied to excitatory and suppressive responses at each measurement site. We found that the superset of CRF features yielded spatially and temporally distributed, generalizable representations of a conspecific song. Individual sites responded to acoustically diverse features, as there was no discernable organization of features across anatomically ordered sites. The CRF features at each site yielded broad, temporally distributed responses that spanned the entire duration of many starling songs, which can last for 50 s or more. Based on these results, we estimated that a nearly complete representation of any conspecific song, regardless of length, can be obtained by evaluating populations as small as 100 neurons. We conclude that natural acoustic communication signals drive a distributed yet highly redundant representation across the songbird auditory forebrain, in which adjacent neurons contribute to the encoding of multiple diverse and time-varying spectro-temporal features.

EEGs Disclose Significant Brain Activity Correlated with Synaptic Fickleness

We here study a network of synaptic relations mingling excitatory and inhibitory neuron nodes that displays oscillations quite similar to electroencephalogram (EEG) brain waves, and identify abrupt variations brought about by swift synaptic mediations. We thus conclude that corresponding changes in EEG series surely come from the slowdown of the activity in neuron populations due to synaptic restrictions. The latter happens to generate an imbalance between excitation and inhibition causing a quick explosive increase of excitatory activity, which turns out to be a (first-order) transition among dynamic mental phases. Moreover, near this phase transition, our model system exhibits waves with a strong component in the so-called delta-theta domain that coexist with fast oscillations. These findings provide a simple explanation for the observed delta-gamma and theta-gamma modulation in actual brains, and open a serious and versatile path to understand deeply large amounts of apparently erratic, easily accessible brain data.

Selective Neuron Vulnerability in Common and Rare Diseases—Mitochondria in the Focus

Mitochondrial dysfunction is a central feature of neurodegeneration within the central and peripheral nervous system, highlighting a strong dependence on proper mitochondrial function of neurons with especially high energy consumptions. The fitness of mitochondria critically depends on preservation of distinct processes, including the maintenance of their own genome, mitochondrial dynamics, quality control, and Ca2+ handling. These processes appear to be differently affected in common neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, as well as in rare neurological disorders, including Huntington’s disease, Amyotrophic Lateral Sclerosis and peripheral neuropathies. Strikingly, particular neuron populations of different morphology and function perish in these diseases, suggesting that cell-type specific factors contribute to the vulnerability to distinct mitochondrial defects. Here we review the disruption of mitochondrial processes in common as well as in rare neurological disorders and its impact on selective neurodegeneration. Understanding discrepancies and commonalities regarding mitochondrial dysfunction as well as individual neuronal demands will help to design new targets and to make use of already established treatments in order to improve treatment of these diseases.

Optogenetic stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsin variants yield opposite behavioral outcomes

Optogenetic manipulation of genetically-specified neuron populations has become a key tool in circuit neuroscience. The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors; interestingly, this amygdalar structure contains a majority of GABAergic projection neurons. A previous study showed that optogenetic activation of MeA GABA neurons with channelrhodopsin-2H134R (ChR2) strongly enhanced inter-male aggression (Hong et al. 2014, Cell). When we attempted to reproduce these findings, accidentally using a faster channelrhodopsin variant (channelrhodopsin-2H134R,E123T or ChETA), we found the opposite results. We therefore systematically compared the behavioral outcome of optogenetic stimulation of MeApd GABA neurons with ChETA versus ChR2, employing two widely used AAV serotypes. This revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression. Recordings of membrane potential changes following optogenetic stimulation with ChETA versus ChR2 revealed larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition of neighboring inhibitory neurons with ChR2 as compared to ChETA. Our study shows that channelrhodopsin variants have to be chosen with care for in-vivo optogenetic experiments. Furthermore, the role of MeApd GABA neurons in aggression control should be re-evaluated.