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B104 cells (clone A) stably transfected with the gene construct depicted in Fig. 1 are shown before (A) and after (B and C) stimulation with 8 nM TPA. Cells were stained with X-Gal to visualize f8-galactosidase activity. Note single cells expressing the fusion gene even without stimulation (A). P-Galactosidase activity is confined to the cell nucleus and shows a distinctive nuclear staining pattern with the nucleoli remaining unstained (arrow in C). (Bar = 50 ,um in A and B and 10 k.m in C.)
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Expression of the c-fos protooncogene is induced by a great variety of extracellular stimuli. A fos-lacZ fusion gene has been constructed that recapitulates this regulation. The fos-lacZ gene was introduced into B104 neuroblastoma cells for use in a quantitative assay for stimulus-transcription coupling. Both alpha- and beta-adrenergic agonists, di...
Citations
... Lastly, the observation that immature MLIs were found expressing rather high levels of immediate early genes, notably Fos (Kozareva et al. 2021; this finding is also supported by the data reported by Vladoiu et al. 2019), is consistent with the significance of external stimuli operative during the development and settling of these cells. However, it should be stressed that the sensitivity of Fos to a broad spectrum of external stimuli (e.g., Schilling et al. 1991b;Smeyne et al. 1992) does not allow us to draw any conclusions as to the nature of those acting on developing MLIs, which indeed may include stimuli due to tissue preparation. ...
The present review aims to provide a short update of our understanding of the inhibitory interneurons of the cerebellum. While these cells constitute but a minority of all cerebellar neurons, their functional significance is increasingly being recognized. For one, inhibitory interneurons of the cerebellar cortex are now known to constitute a clearly more diverse group than their traditional grouping as stellate, basket, and Golgi cells suggests, and this diversity is now substantiated by single-cell genetic data. The past decade or so has also provided important information about interneurons in cerebellar nuclei. Significantly, developmental studies have revealed that the specification and formation of cerebellar inhibitory interneurons fundamentally differ from, say, the cortical interneurons, and define a mode of diversification critically dependent on spatiotemporally patterned external signals. Last, but not least, in the past years, dysfunction of cerebellar inhibitory interneurons could also be linked with clinically defined deficits. I hope that this review, however fragmentary, may stimulate interest and help focus research towards understanding the cerebellum.
... We focused on the postnatal day 6 (P6), at the time window when USV features show a significant difference between pup groups derived from young and aged fathers in our previous research. 13,16 We utilized the expression of c-Fos, a protein product of the neuronal immediate-early genes (IEGs) c-Fos, 17 which has been well characterized and widely used as a marker to facilitate neuronal activity mapping on histological brain sections. [18][19][20] First, we performed fluorescence immunostaining using anti-c-Fos antibody after maternal separation in a series of brain sections across the entire mouse brain. ...
Neonatal mice emit ultrasonic vocalizations (USVs) when separated from their mothers. Since the USVs attract their mothers' attention and trigger maternal retrieval, they are considered to serve as social signals for communication. We have modeled paternal aging effects on the vocal communication of offspring in mice. However, little is known about the neural basis underlying neonatal USV production. To identify responsible brain regions driving the vocal behavior, we comprehensively mapped the neuronal activity associated with USV production in the entire brain of mice at postnatal day 6 (P6). Using an expression of immediate-early gene c-Fos as a neuronal activity marker, correlations between the numbers of USVs and c-Fos positive neurons were analyzed. We identified 23 candidate brain regions associated with USV production in the mice at P6. Our study would be a first step toward comprehensively understanding the neuronal mechanisms that regulate and develop vocal behaviors in neonatal mice.
... Furthermore, c-fos can negatively regulate its own expression and this characteristic is required for a rapid decline in its expression . In neurons, the first detailed studies assessing the regulatory mechanisms of IEGs were performed on c-fos (Schilling et al. 1991;Okuno 2011) and, under baseline conditions, there is little or no expression of this gene in most neurons (Morgan and Curran 1989;Hoffman et al. 1993). The expression of various late-response genes involved in different neuronal processes (for example growth control or plastic changes) is induced by the activation of c-fos gene (Sukhatme et al. 1988;Williams et al. 2000;Bozon et al. 2003;Maddox et al. 2011;Gallo et al. 2018). ...
Over the last few decades, it has been shown that fish, comprising the largest group of vertebrates and in many respects one of the least well studied, possess many cognitive abilities comparable to those of birds and mammals. Despite a plethora of behavioural studies assessing cognition abilities and an abundance of neuroanatomical studies, only few studies have aimed to or in fact identified the neural substrates involved in the processing of cognitive information. In this review, an overview of the currently available studies addressing the joint research topics of cognitive behaviour and neuroscience in teleosts (and elasmobranchs wherever possible) is provided, primarily focusing on two fundamentally different but complementary approaches, i.e. ablation studies and Immediate Early Gene (IEG) analyses. More recently, the latter technique has become one of the most promising methods to visualize neuronal populations activated in specific brain areas, both during a variety of cognitive as well as non-cognition-related tasks. While IEG studies may be more elegant and potentially easier to conduct, only lesion studies can help researchers find out what information animals can learn or recall prior to and following ablation of a particular brain area.
... A common strategy adopted to label activated neuronal ensembles takes advantage of enhancer and promoter sequences of activity-dependent genes whose transcription is activated upon plasticity-inducing stimuli (Kawashima et al., 2014;DeNardo and Luo, 2017;Josselyn and Tonegawa, 2020). The promoters most widely utilized to aid neuronal activity mapping are those of the Fos, Erg1, and Arc/Arg3.1 genes (Schilling et al., 1991;Waltereit et al., 2001;Kawashima et al., 2009;Pintchovski et al., 2009;Guenthner et al., 2013;DeNardo and Luo, 2017;Sauvage et al., 2019). Alternatively, the recently developed RAM system allows for active ensemble neuron labeling thanks to a synthetic promoter which combines the binding sites for the activity-dependent transcription factors Fos and Npas4 (Sørensen et al., 2016). ...
Increasing evidence supports a model whereby memories are encoded by sparse ensembles of neurons called engrams, activated during memory encoding and reactivated upon recall. An engram consists of a network of cells that undergo long-lasting modifications of their transcriptional programs and connectivity. Ground-breaking advancements in this field have been made possible by the creative exploitation of the characteristic transcriptional responses of neurons to activity, allowing both engram labeling and manipulation. Nevertheless, numerous aspects of engram cell-type composition and function remain to be addressed. As recent transcriptomic studies have revealed, memory encoding induces persistent transcriptional and functional changes in a plethora of neuronal subtypes and non-neuronal cells, including glutamatergic excitatory neurons, GABAergic inhibitory neurons, and glia cells. Dissecting the contribution of these different cellular classes to memory engram formation and activity is quite a challenging yet essential endeavor. In this review, we focus on the role played by the GABAergic inhibitory component of the engram through two complementary lenses. On one hand, we report on available physiological evidence addressing the involvement of inhibitory neurons to different stages of memory formation, consolidation, storage and recall. On the other, we capitalize on a growing number of transcriptomic studies that profile the transcriptional response of inhibitory neurons to activity, revealing important clues on their potential involvement in learning and memory processes. The picture that emerges suggests that inhibitory neurons are an essential component of the engram, likely involved in engram allocation, in tuning engram excitation and in storing the memory trace.
... Several transgenic mouse lines exist. At first, the promoter of c-Fos was isolated and fused with beta-galactosidase, generating Fos-lacZ transgenic mouse line (Schilling et al., 1991), enabling β-gal staining of activated cells. Subsequently other IEG were coupled to lacZ expression to generate transgenic mouse line. ...
La formation de nouveaux souvenirs après une expérience unique nécessite un encodage initial rapide puis une consolidation dans le temps. Pendant l'apprentissage, les informations multimodales convergent vers l'hippocampe, activant des assemblages neuronaux épars. On pense que les neurones activés forment une représentation de la mémoire par une activité concertée et une interconnectivité synaptique. Des études computationnelles et comportementales montrent que la région CA3 de l'hippocampe est une structure clé impliquée dans l'intégration d'informations multimodales et le stockage rapide de la mémoire. Dans cette étude, les propriétés des neurones CA3 activés et de leur réseau local après l'apprentissage en une seule fois d'une mémoire de type épisodique sont explorées. Des constructions virales basées sur le gène immédiatement précoce cFos sont utilisées pour identifier et suivre ex vivo les assemblages CA3 à différents moments de leur émergence et de leur développement. Nous décrivons l'utilisation d'une nouvelle construction basée sur le cFos permettant l'expression transitoire dépendante de l'activité d'un marqueur fluorescent déstabilisé ZsGreen rapidement après un apprentissage d'essai (quelques heures). En parallèle, le système de marqueurs d'activité robustes (RAM), qui permet un marquage dépendant de l'activité 24 heures après une expérience inédite, est utilisé pour caractériser les assemblages neuronaux après une phase initiale de consolidation. Les propriétés électrophysiologiques des cellules pyramidales CA3 marquées avec ces techniques sont rapportées au moyen d'enregistrements par patch-clamp dans des tranches hippocampiques ex vivo. Nous analysons les propriétés intrinsèques et la connectivité synaptique des neurones CA3 activés par une tâche de mémoire à un essai, en comparaison avec les neurones pyramidaux voisins non activés, ce qui fournit des informations sur les changements de ces propriétés au cours de la période initiale d'encodage et de la première phase de consolidation.
... The c-fos-lacZ transgenic rats were generated by James I. Morgan's laboratory about 25 years ago. The scientists microinjected the c-fos-lacZ transgene (Fig. 14), which was previously described by Schilling et al. (1991), into the pronuclei of fertilized ova of female Sprague-Dawley rats (Kasof et al., 1995;Koya et al., 2009b). Transgenic c-fos-lacZ rats transcribe the lacZ coding sequence under control of the c-fos promotor, resulting in translation into the protein ß-galactosidase in strongly activated neurons (Cruz et al., 2013). ...
... The 3' untranslated region of c-fos furthermore contains the SV40 polyadenylation signal (pA). Adapted and modified from (Schilling et al., 1991). ...
Our daily life activities are highly dependent on rewards and their ability to drive our motivational and goal-directed behaviours. Rewards are essential for all individuals as they enable us to satisfy our most basic needs and desires. The processing of exteroceptive and interoceptive stimuli within the reward system help us to evaluate the rewarding value of certain actions and reward entities. In this regard, the reward system is originally responsible for processing of information related to natural rewards including food, water and sex. However, it can also be hijacked by drugs of abuse, which are able to manipulate the system with their reinforcing properties. Drugs of abuse are thereby able to drive learning processes, as a result of which external stimuli, such as odours, sounds or visual cues, become associated with the respective drug. For addicts, exposure to these stimuli triggers drug seeking, which results in drug consumption and, for abstinent individuals, relapse of compulsive drug use. In order to improve relapse prevention with pharmacological or therapeutical interventions the underlying mechanisms must be understood on a neuronal level. Today, it is widely accepted that distinct memories, such as learned cue-reward associations, are encoded in sparsely distributed subsets of neurons, so called neuronal ensembles, to achieve given tasks. Rewarding stimuli are thereby thought to activate distinct neurobiological patterns within the reward system. Thus, the associative learning process might result in the formation of neuronal ensembles that encode cue-reward associations, which are reward specific. This thesis therefore aimed to examine the shared and distinct properties of neuronal ensembles encoding different cue-reward associations, investigating different drugs of abuse and the natural reward saccharin. In a first set of experiments, we identified co-active neurons encoding for the investigated cue-induced reward seeking behaviour within the extended reward system. The natural reward saccharin as well as the three drugs of abuse (ethanol, cocaine, nicotine) showed broadly overlapping neuronal activity patterns within the prefrontal cortex, specifically within its orbitofrontal part. On a subcortical level similar activation patterns could be found within the caudate putamen. Despite these similarities, each reward also displayed distinct changes in neuronal activity. Cue-induced ethanol seeking, for example, increased activity within the basolateral amygdala, whereas cue-induced seeking for both psychostimulants displayed elevated activity within the nucleus accumbens core. Saccharin trained animals, however, showed activity reduction within the ventral tegmental area. A second set of experiments characterized the previously identified ensembles on a neurochemical level. The focus was thereby put on ensembles activated within the orbitofrontal cortex, the brain region with the highest activity during cue-induced reward seeking. Immunohistochemical co-localizations showed that all ensembles activated during reward seeking were entirely neuronal and did not comprise any neuroglia. Most remarkably however was the finding that neuronal ensembles within the orbitofrontal cortex displayed a reward specific neurochemical composition. Whereas the neuronal ensemble activated by cue-induced saccharin seeking showed a balanced participation of glutamatergic and GABAergic neurons, the ensemble induced by ethanol seeking was primarily GABAergic and the ensemble triggered by cocaine seeking was predominantly glutamate driven. In a further experiment, the previously identified neuronal ensembles within the orbitofrontal cortex were functionally validated for their involvement in cue-induced saccharin seeking or cue-induced cocaine seeking behaviour. It was found that inactivation of neuronal ensembles responsible for cue-induced saccharin seeking within the orbitofrontal cortex had an impact on reward seeking behaviour whereas abolition of neuronal ensembles involved in cue-induced cocaine seeking showed no effect on the behavioural output. Finally, neuronal ensembles within the infralimbic cortex mediating ethanol and saccharin seeking were investigated. Examination of participating neurons revealed that both ensembles were largely overlapping but also displayed distinct components specific to each reward. Overall, the present work contributes to a better understanding of the organisation and functionality of neuronal ensembles involved in cue-induced reward seeking behaviour. The discovered reward dependent neurochemical distinctness of neuronal ensembles encoding cue-reward associations may provide the opportunity for new therapeutical approaches focusing on the distinct features. However, further studies are necessary to further clarify the role of these distinct reward specific components. Nevertheless, the findings of this thesis provide a good starting point to gain more knowledge about neuronal ensembles and their underlying mechanisms and how these might be addressed in a drug-specific manner during addiction treatment.
... Although the SCN is most electrically active during the day (Colwell, 2011), for the SCN clock to control nighttime activity we hypothesized, some SCN neurons would be electrically active during one or more of these nighttime events. We therefore quantified SCN neuron electrical activity at six time points by immunostaining for the immediate-early protein c-FOS, a marker of neural activity (Schilling et al., 1991) (Figure 1C). Consistent with previous reports, significantly more neurons express c-FOS during the day than during the night (Colwell, 2011). ...
Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle.
... To specifically label activated OT neurons, we developed and describe here a genetic method called vGATE ( Figure 1A). In the vGATE system, a c-fos promoter (P fos ) fragment ( Schilling et al., 1991) drives the expression of the reverse tetracyclinesensitive (tet) transactivator (rtTA) ( Dogbevia et al., 2015Dogbevia et al., , 2016. In activated neurons, stimulation of the c-fos promoter rapidly induces rtTA expression. ...
... The vGATE system Generation and cloning We engineered a synthetic c-fos promoter linked to its first exon (Exon 1; ( Schilling et al., 1991) with ATGs in the Exon 1 converted to TTGs by site directed mutagenesis and heptamerized tetracycline (tet) operators, (tetO) 7 , added upstream of it. These operators drive the expression of a humanized reverse tet transactivator (rtTA) ( Dogbevia et al., 2015) named (tetO) 7 -P fos -rtTA. ...
Oxytocin (OT) release by axonal terminals onto the central nucleus of the amygdala exerts anxiolysis. To investigate which subpopulation of OT neurons contributes to this effect, we developed a novel method: virus-delivered genetic activity-induced tagging of cell ensembles (vGATE). With the vGATE method, we identified and permanently tagged a small subpopulation of OT cells, which, by optogenetic stimulation, strongly attenuated contextual fear-induced freezing, and pharmacogenetic silencing of tagged OT neurons impaired context-specific fear extinction, demonstrating that the tagged OT neurons are sufficient and necessary, respectively, to control contextual fear. Intriguingly, OT cell terminals of fear-experienced rats displayed enhanced glutamate release in the amygdala. Furthermore, rats exposed to another round of fear conditioning displayed 5-fold more activated magnocellular OT neurons in a novel environment than a familiar one, possibly for a generalized fear response. Thus, our results provide first evidence that hypothalamic OT neurons represent a fear memory engram.
... While descriptive methods such as proteomics and transcriptomics have delivered insight into the synaptic architecture and neuronal activity-dependent gene expression, respectively 8,18,19 , the empirical association of gene functions with a dedicated neuronal phenotype is a tedious endeavour and high-throughput techniques are not yet available. The activity state of neurons has long been visualized using reporter proteins controlled by activity-dependent promoters or enhancer elements [20][21][22][23] . We and others have demonstrated that signalling can be robustly measured by deep sequencing using pathway-specific reporters expressing short synthetic RNA barcode sequences 24,25 . ...
Abstract Neuronal signal transduction shapes brain function and malfunction may cause mental disorders. Despite the existence of functional genomics screens for proliferation and toxicity, neuronal signalling has been difficult to address so far. To overcome this limitation, we developed a pooled screening assay which combines barcoded activity reporters with pooled genetic perturbation in a dual-expression adeno-associated virus (AAV) library. With this approach, termed pathScreener, we comprehensively dissect signalling pathways in postmitotic neurons. This overcomes several limitations of lentiviral-based screens. By applying first a barcoded and multiplexed reporter assay, termed cisProfiler, we identified the synaptic-activity responsive element (SARE) as top performance sensor of neuronal activity. Next, we targeted more than 4,400 genes and screened for modulatory effects on SARE activity in primary cortical neurons. We identified with high replicability many known genes involved in glutamatergic synapse-to-nucleus signalling of which a subset was validated in orthogonal assays. Several others have not yet been associated with the regulation of neuronal activity such as the hedgehog signalling members Ptch2 and Ift57. This assay thus enhances the toolbox for analysing regulatory processes during neuronal signalling and may help identifying novel targets for brain disorders.
... Male cFos-lacZ transgenic rats (Kasof et al., 1995;Koya et al., 2009), carrying a cFos-lacZ fusion protein (Schilling et al., 1991) Male Indiana alcohol preferring (P) and non-preferring (NP) rats (Lumeng et al., 1977) were obtained from the Animal Production Core facility at the Indiana Alcohol Research ...
The medial prefrontal cortex (mPFC) is critically involved in cognitive flexibility and top down control of behavior. Dysfunction of this brain region is a hallmark of many psychiatric disorders including addiction. The physiological and molecular mechanisms underlying mPFC function are largely unclear. A widely accepted theory posits that distinct memories are encoded in the brain by sparsely distributed sets of neurons, so called neuronal ensembles, which has been demonstrated for reward seeking behavior. However, in the case of alcohol seeking neuronal ensembles had not been identified and it is unclear how such ensembles might differ from those involved in natural reward seeking. Furthermore, excessive alcohol use causes damage to the mPFC, especially to its ventromedial subregion, also termed infralimbic cortex (IL). Long-term alcohol-induced changes in this brain area include a deficit in metabotropic glutamate receptor subtype 2 (mGluR2). These receptors modify the signaling properties of IL neurons to their projection targets and their dysfunction within corticostriatal projections of alcohol-dependent rats is known to be associated with loss of control over alcohol seeking behavior. Thus, this PhD thesis aims to provide insights into the organization of IL neuronal ensembles involved in alcohol and natural reward seeking and to further understand the role of an mGluR2 deficit for IL dependent control over alcohol seeking and cognitive flexibility. In Study 1 we identify a functional neuronal ensemble in the IL involved in the control of alcohol seeking behavior, using a chemo-genetic inactivation method. In Study 2 we demonstrate that IL neuronal ensembles involved in alcohol and saccharin seeking are highly overlapping, but also contain reward specific components by using retrograde tracing techniques in combination with a novel two-reward operant task. In Study 3 we develop an advanced methodological framework for measuring neuronal ensemble activity during an operant reward seeking task using in-vivo calcium imaging. By using viral mGluR2 knockdown techniques, Study 4 and 5 establish an IL mGluR2 deficit as a common pathological mechanism for excessive alcohol seeking and impaired cognitive flexibility. In summary, the results of this thesis provide important insights into the function and organization of neuronal ensembles involved in reward seeking. Possible changes of organization and function of neuronal ensembles in pathological conditions, like addiction, should be addressed in future studies. Furthermore an IL mGluR2 deficit is established as a common pathological mechanism for excessive alcohol seeking and impaired cognitive flexibility, thus leading to a deeper understanding of the underlying molecular mechanisms of this frequent comorbidity and providing a promising target for future medication therapies.
