Compartment Analysis Of Temporal Activity Research Articles

Estrous cycle state-dependent renewal of appetitive behavior recruits unique patterns of Arc mRNA in female rats.

Renewal is a behavioral phenomenon wherein extinction learning fails to generalize between different contextual environments, thereby representing a significant challenge to extinction-based rehabilitative therapies. Previously, we have shown that renewal of extinguished appetitive behavior differs across the estrous cycle of the female rat. In this experiment that effect is replicated and extended upon to understand how the estrous cycle may modulate contextual representation at the neuronal population level to drive renewal. Estrous cycle stage [i.e., proestrus (P, high hormone) or metestrus/diestrus (M/D, low hormone)] was considered during two important learning and behavioral expression windows: at extinction training and during long-term memory (LTM)/renewal testing. Cellular compartment analysis of temporal activity using fluorescence in situ hybridization (catFISH) for Arc mRNA was conducted after the distinct context-stimulus exposures. Rats in P during context-dependent extinction training but in a different stage of the estrous cycle during LTM and renewal testing (P-different) were shown to exhibit more renewal of conditioned foodcup (but not conditioned orienting) behavior compared to rats in other estrous cycle groups. Importantly, we discovered this depends on the order of tests. P-different rats showed differential Arc mRNA expression in regions of the prefrontal cortex (PFC), amygdala, and hippocampus (HPC). For each case P-different rats had more co-expression (i.e., expression of both nuclear and cytoplasmic) of Arc mRNA compared to other groups; specific to the dorsal HPC, P-different rats also had a more robust Arc mRNA response to the extinction context exposure. These data suggest female rats show estrous cycle state-dependent renewal of appetitive behavior, and differences in context and conditioned stimulus representation at the neuronal level may drive this effect.

Behavioral and Cellular Tagging in Young and in Early Cognitive Aging.

The ability to maintain relevant information on a daily basis is negatively impacted by aging. However, the neuronal mechanism manifesting memory persistence in young animals and memory decline in early aging is not fully understood. A novel event, when introduced around encoding of an everyday memory task, can facilitate memory persistence in young age but not in early aging. Here, we investigated in male rats how sub-regions of the hippocampus are involved in memory representation in behavioral tagging and how early aging affects such representation by combining behavioral training in appetitive delayed-matching-to-place tasks with the “cellular compartment analysis of temporal activity by fluorescence in situ hybridization” technique. We show that neuronal assemblies activated by memory encoding were also partially activated by novelty, particularly in the distal CA1 and proximal CA3 subregions in young male rats. In early aging, both encoding- and novelty-triggered neuronal populations were significantly reduced with a more profound effect in encoding neurons. Thus, memory persistence through novelty facilitation engages overlapping hippocampal assemblies as a key cellular signature, and cognitive aging is associated with underlying reduction in neuronal activation.

Plasticity-Related Activity in the Hippocampus, Anterior Cingulate, Orbitofrontal, and Prefrontal Cortex Following a Repeated Treatment with D2/D3 Agonist Quinpirole.

Quinpirole (QNP) sensitization is a well-established model of stereotypical checking relevant to obsessive-compulsive disorder. Previously, we found that QNP-treated rats display deficits in hippocampus-dependent tasks. The present study explores the expression of immediate early genes (IEG) during QNP-induced stereotypical checking in the hippocampus, anterior cingulate cortex (ACC), orbitofrontal cortex (OFC), and medial prefrontal cortex (mPFC). Adult male rats were injected with QNP (0.5 mg/mL/kg; n = 15) or saline (n = 14) daily for 10 days and exposed to an arena enriched with two objects. Visits to the objects and the corners of the arena were recorded. QNP-treated rats developed an idiosyncratic pattern of visits that persisted across experimental days. On day 11, rats were exposed to the arena twice for 5 min and sacrificed. The expression of IEGs Arc and Homer1a was determined using cellular compartment analysis of temporal activity by fluorescence in situ hybridization. IEG-positive nuclei were counted in the CA1 area of the hippocampus, ACC, OFC, and mPFC. We found significantly fewer IEG-positive nuclei in the CA1 in QNP-treated rats compared to controls. The overlap between IEG expressing neurons was comparable between the groups. We did not observe significant differences in IEG expression between QNP treated and control rats in ACC, OFC, and mPFC. In conclusion, treatment of rats with quinpirole decreases plasticity-related activity in the hippocampus during stereotypical checking.

Distinct Populations of Neurons Activated by Heroin and Cocaine in the Striatum as Assessed by catFISH.

Despite the still prevailing notion of a shared substrate of action for all addictive drugs, there is evidence suggesting that opioid and psychostimulant drugs differ substantially in terms of their neurobiological and behavioral effects. These differences may reflect separate neural circuits engaged by the two drugs. Here we used the catFISH (cellular compartment analysis of temporal activity by fluorescence in situ hybridization) technique to investigate the degree of overlap between neurons engaged by heroin versus cocaine in adult male Sprague Dawley rats. The catFISH technique is a within-subject procedure that takes advantage of the different transcriptional time course of the immediate-early genes homer 1a and arc to determine to what extent two stimuli separated by an interval of 25 min engage the same neuronal population. We found that throughout the striatal complex the neuronal populations activated by noncontingent intravenous injections of cocaine (800 μg/kg) and heroin (100 and 200 μg/kg), administered at an interval of 25 min from each other, overlapped to a much lesser extent than in the case of two injections of cocaine (800 μg/kg), also 25 min apart. The greatest reduction in overlap between populations activated by cocaine and heroin was in the dorsomedial and dorsolateral striatum (∼30% and ∼22%, respectively, of the overlap observed for the sequence cocaine-cocaine). Our results point toward a significant separation between neuronal populations activated by heroin and cocaine in the striatal complex. We propose that our findings are a proof of concept that these two drugs are encoded differently in a brain area believed to be a common neurobiological substrate to drug abuse.

Reactivation of Recall-Induced Neurons in the Infralimbic Cortex and the Basolateral Amygdala After Remote Fear Memory Attenuation.

Whether the attenuation of traumatic memories is mediated through the suppression of the original memory trace of fear by a new memory trace of safety, or through an updating of the original fear trace towards safety has been a long-standing question at the interface of neuroscience and psychology. This matter is of particular importance for remote fear memories as they lie at the core of stress- and anxiety-related disorders. Recently, we have found that in the dentate gyrus, the effective attenuation of remote fear memories is accompanied by a reactivation of memory recall-induced neurons and that the continued activity of these neurons is critical for fear reduction. However, whether this also applies to other brain areas implicated in the storage of remote fear memories remains to be determined. Here, we show—by cellular compartment analysis of temporal activity using fluorescence in situ hybridization—that such reactivation also occurs in the basolateral amygdala and the infralimbic cortex, two brain areas known to be involved in fear memory attenuation. These results provide further experimental support for effective traumatic memory attenuation likely being mediated by an updating of the original fear trace towards safety.

New Tools in Cognitive Neurobiology: Biotin-Digoxigenin Detection of Overlapping Active Neuronal Populations by Two-Color c-fos Compartment Analysis of Temporal Activity by Fluorescent in situ Hybridization (catFISH)

The method of cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH) is widely used in cellular and behavioral neurobiology. It exploits stimulus-induced expression of immediate early genes (IEGs) and allows identification of two neuronal populations activated in the brain of the same animal in response to neural or behavioral events spaced by 25-30 min. The differential labeling is based on visualization of nuclear RNA and cytoplasmic mRNA which is accumulated in neurons at different intervals after their activation. However, the drawback of this method is that the two RNA forms are labeled by the same color which complicates their discrimination during image analysis. Methods: We describe a protocol for the two-color c-fos catFISH that allows visualization of the cytoplasmic (stimulus 1) and nuclear (stimulus 2) RNA not only by their intracellular localization but also by the color-coded labeling of spliced and unspliced forms of c-fos RNA using the combination of digoxigenin- and biotin-conjugated probes. We also demonstrate possibility of combining this protocol with immunofluorescent detection of c-Fos protein, which allows visualization of two neuronal populations activated by stimuli spaced from 30 to 60 min. Results: We used the developed protocol for two-color c-fos catFISH to identify neuronal populations activated in the mouse brain by two cognitive stimuli. We identified two color signals that corresponded to cytoplasmic and nuclear c-fos RNA in different brain regions after mice were exposed to two different contexts separated by 20 minutes. Two-color c-fos catFISH protocol was also combined with с-Fos immunohistochemistry, allowing colocalization of c-fos mRNA and с-Fos protein activated by two stimuli spaced by 60 min. Conclusions: The combination of the proposed approaches allows visualization of neuronal populations activated in the brain of the same animal during two or more cognitive episodes in the time window of 60-120 min.

Age-related Changes in Lateral Entorhinal and CA3 Neuron Allocation Predict Poor Performance on Object Discrimination

Age-related memory deficits correlate with dysfunction in the CA3 subregion of the hippocampus, which includes both hyperactivity and overly rigid activity patterns. While changes in intrinsic membrane currents and interneuron alterations are involved in this process, it is not known whether alterations in afferent input to CA3 also contribute. Neurons in layer II of the lateral entorhinal cortex (LEC) project directly to CA3 through the perforant path, but no data are available regarding the effects of advanced age on LEC activity and whether these activity patterns update in response to environmental change. Furthermore, it is not known the extent to which age-related deficits in sensory discrimination relate to the inability of aged CA3 neurons to update in response to new environments. Young and aged rats were pre-characterized on a LEGO© object discrimination task, comparable to behavioral tests in humans in which CA3 hyperactivity has been linked to impairments. The cellular compartment analysis of temporal activity with fluorescence in situ hybridization for the immediate-early gene Arc was then used to identify the principal cell populations that were active during two distinct epochs of random foraging in different environments. This approach enabled the extent to which rats could discriminate two similar objects to be related to the ability of CA3 neurons to update across different environments. In both young and aged rats, there were animals that performed poorly on the LEGO object discrimination task. In the aged rats only, however, the poor performers had a higher percent of CA3 neurons that were active during random foraging in a novel environment, but this is not related to the ability of CA3 neurons to remap when the environment changed. Afferent neurons to CA3 in LEC, as identified with the retrograde tracer choleratoxin B (CTB), also showed a higher percentage of cells that were positive for Arc mRNA in aged poor performing rats. This suggests that LEC contributes to the hyperactivity seen in CA3 of aged animals with object discrimination deficits and age-related cognitive decline may be the consequence of dysfunction endemic to the larger network.

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5–8 consecutive burst-induced nuclear calcium transients at 0.05–0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased.

Extinction and Retrieval + Extinction of Conditioned Fear Differentially Activate Medial Prefrontal Cortex and Amygdala in Rats.

Pairing a previously neutral conditioned stimulus (CS; e.g., a tone) to an aversive unconditioned stimulus (US; e.g., a footshock) leads to associative learning such that the tone alone comes to elicit a conditioned response (e.g., freezing). We have previously shown that an extinction session that occurs within the reconsolidation window (termed retrieval + extinction) attenuates fear responding and prevents the return of fear in Pavlovian fear conditioning (Monfils et al., 2009). To date, the mechanisms that explain the different behavioral outcomes between standard extinction and retrieval + extinction remain poorly understood. Here we sought to examine the differential temporal engagement of specific neural systems by these two approaches using Arc catFISH (cellular compartment analysis of temporal activity using fluorescence in situ hybridization (FISH)). Our results demonstrate that extinction and retrieval + extinction lead to differential patterns of expression, suggesting that they engage different networks. These findings provide insight into the neural mechanisms that allow extinction during reconsolidation to prevent the return of fear in rodents.

Influence of Isoflurane on Immediate-Early Gene Expression.

Background: Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration.Methods: Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 h later for long-term memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH), we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex.Results: On a cellular level, isoflurane administered at high doses (general anesthesia) prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training.Conclusion: General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the effects of isoflurane on memory formation. Combined with published research on the effects of volatile anesthetics on memory in behaving animals, our data suggests that different levels of anesthesia affect memory via different mechanisms: general anesthesia prevents elevation of mRNA levels of Arc and Zif268 which are necessary for normal memory formation, while anesthesia at lower doses affects the strength of memory by affecting levels of plasticity-related proteins.

Artificial association of pre-stored information to generate a qualitatively new memory.

Memory is thought to be stored in the brain as anensemble of cells activated during learning. Although optical stimulation of a cell ensemble triggers the retrieval of the corresponding memory, it is unclear how the association of information occurs at the cell ensemble level. Using optogenetic stimulation without any sensory input in mice, we found that an artificial association between stored, non-related contextual, and fear information was generated through the synchronous activation of distinct cell ensembles corresponding to the stored information. This artificial association shared characteristics with physiologically associated memories, such as N-methyl-D-aspartate receptor activity and protein synthesis dependence. These findings suggest that the association of information is achieved through the synchronous activity of distinct cell ensembles. This mechanism may underlie memory updating by incorporating novel information into pre-existing networks to form qualitatively new memories.

Neuronal ensembles in amygdala, hippocampus, and prefrontal cortex track differential components of contextual fear.

Although the circuit mediating contextual fear conditioning has been extensively described, the precise contribution that specific anatomical nodes make to behavior has not been fully elucidated. To clarify the roles of the dorsal hippocampus (DH), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) in contextual fear conditioning, activity within these regions was mapped using cellular compartment analysis of temporal activity using fluorescence in situ hybridization (catFISH) for Arc mRNA. Rats were delay-fear conditioned or immediately shocked (controls) and thereafter reexposed to the shocked context to test for fear memory recall. Subsequent catFISH analyses revealed that in the DH, cells were preferentially reactivated during the context test, regardless of whether animals had been fear conditioned or immediately shocked, suggesting that the DH encodes spatial information specifically, rather then the emotional valence of an environment. In direct contrast, neuronal ensembles in the BLA were only reactivated at test if animals had been fear conditioned, suggesting that the amygdala specifically tracks the emotional properties of a context. Interestingly, Arc expression in the mPFC was consistent with both amygdala- and hippocampus-like patterns, supporting a role for the mPFC in both fear and contextual processing. Collectively, these data provide crucial insight into the region-specific behavior of neuronal ensembles during contextual fear conditioning and demonstrate a dissociable role for the hippocampus and amygdala in processing the contextual and emotional properties of a fear memory.

Ensemble coding of context-dependent fear memory in the amygdala.

After fear conditioning, presenting the conditioned stimulus (CS) alone yields a context-specific extinction memory; fear is suppressed in the extinction context, but renews in any other context. The context-dependence of extinction is mediated by a brain circuit consisting of the hippocampus, prefrontal cortex (PFC) and amygdala. In the present work, we sought to determine at what level of this circuit context-dependent representations of the CS emerge. To explore this question, we used cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH). This method exploits the intracellular expression profile of the immediate early gene (IEG), Arc, to visualize neuronal activation patterns to two different behavioral experiences. Rats were fear conditioned in one context and extinguished in another; 24 h later, they were sequentially exposed to the CS in the extinction context and another context. Control rats were also tested in each context, but were never extinguished. We assessed Arc mRNA expression within the basal amygdala (BA), lateral amygdala (LA), ventral hippocampus (VH), prelimbic cortex (PL) and infralimbic cortex (IL). We observed that the sequential retention tests induced context-dependent patterns of Arc expression in the BA, LA, and IL of extinguished rats; this was not observed in non-extinguished controls. In general, non-extinguished animals had proportionately greater numbers of non-selective (double-labeled) neurons than extinguished animals. Collectively, these findings suggest that extinction learning results in pattern separation, particularly within the BA, in which unique neuronal ensembles represent fear memories after extinction.

Off‐line Arc transcription in active ensembles during fear memory retrieval

Neural activity and de novo protein synthesis during a rest period following memory retrieval in the amygdala is necessary for stabilization of reactivated fear memory. Arc/Arg3.1 (Arc) expression is regulated by neural activity and is a critical protein for memory reconsolidation. However, it remains unclear whether memory retrieval alters Arc transcription during subsequent rest. In this study, the populations of mouse lateral amygdala neurons that transcribe Arc during memory retrieval and at rest were detected using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (Arc catFISH). Results demonstrated that memory retrieval alters the composition of neuronal populations, which activate Arc transcription during subsequent rest. Approximately 50% of neurons that transcribe Arc at subsequent rest, transcribed Arc during memory retrieval, whereas only approximately 10% of neurons that transcribed Arc during a rest period prior to memory retrieval transcribe Arc during memory retrieval. In contrast, re-exposure to the chamber induced less preferential Arc transcription in latent inhibited mice that received shocks but recalled less conditioned fear. Taken together, these findings indicate that neuronal subpopulations activated during fear memory retrieval preferentially transcribe Arc during subsequent rest in the lateral amygdala. This preferential Arc transcription may contribute to memory reconsolidation.

Population activity in the dorsal hippocampal CA1 encoding the surrounding environment is absent during contextual fear memory expression

The hippocampus plays a critical role in contextual fear conditioning. Population activity in the hippocampal CA1 encoding the surrounding environment is thought to be responsible for retrieval of contextual fear memory. However, the characteristics of CA1 neuronal ensemble activity during retrieval of contextual fear memory remain unclear. Here, we examined CA1 ensemble activity during contextual fear memory expression in male C57Bl/6J mice, using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization. The “Shock” group was conditioned with a footshock in two separate chambers, whereas the “No shock” group was not exposed to shocks in the chamber. Animals were then re-exposed to either the same chamber twice or two different conditioning chambers. In the No shock group, exposure to the same chamber twice activated a more significantly overlapping neuronal population than exposure to two different chambers. In the Shock group, exposure to the same conditioning chamber twice activated a similarly overlapping neuronal population as exposure to two different chambers, with overlap smaller than in nonshocked mice exposed to the same chamber twice. Thus, population activity in the hippocampal CA1 encoding the surrounding environment is detected during spatial exploration, but absent during contextual fear memory expression. Even the variable ensemble activity of CA1 may contribute to retrieval of contextual fear memory.

Preferential Arc transcription at rest in the active ensemble during associative learning

Information processing in the central nervous system (CNS) during periods of rest is crucial for lasting memories but the precise off-line neuronal population activity that contributes to long-term memory formation remains unclear. This pattern of neuronal activity during rest triggers transcription of immediate early genes such as activity regulated cytoskeletal gene (Arc). We compared the active neuronal population in the lateral amygdala of C57BL/6J mice during fear conditioning and rest periods using a large scale imaging technique, Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH). We found that the neuronal population transcribing Arc during fear conditioning was more similar to that the population transcribing Arc after fear conditioning than before fear conditioning. The overlapping population was larger in conditioned mice that acquired associative memory than in unshocked mice and in latent inhibited mice that received shocks but did not form associative memory. Moreover, these results were confirmed using Arc/Homer 1a catFISH. Our findings indicate that Arc is preferentially transcribed in neurons that are active during fear conditioning after associative learning. This preferential transcription may contribute to the formation of long-lasting memory.

Immediate-Early Gene Expression at Rest Recapitulates Recent Experience

Immediate-early genes (IEGs) are tightly coupled to cellular activity and play a critical role in regulating synaptic plasticity. While encoding spatial experience, hippocampal principal cells express IEGs in a behaviorally dependent and cell-specific manner. This expression can be detected through the use of cellular compartment analysis of temporal activity by fluorescence in situ hybridization to generate estimates of cellular activity that match direct neuronal recording under comparable conditions. During rest, IEG expression continues to occur in a small number of cells, and the role of this basal expression is unknown. Imaging IEGs expressed during exploration and adjacent rest periods reveals that "constitutive" IEG expression during rest is not random. Rather, consistent with proposed memory consolidation mechanisms, it recapitulates a subset of the pattern generated by recent experience.

Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches

In the 14 years since it was discovered that specific genes could be dynamically regulated in the brain by neural activity, there has been a substantial research focus attempting to understand the role immediate-early genes (IEGs) play in various brain functions. This article examines the involvement of IEGs in hippocampal synaptic plasticity and in memory consolidation processes performed by the hippocampus. Studies employing conventional IEG detection methodologies and a novel gene-imaging approach that provides temporal and cellular resolution (cellular compartment analysis of temporal activity by fluorescence in situ hybridization or catFISH) provide evidence supporting the assertion that IEG expression reflects the integration of information processed by hippocampal neurons. However, IEG expression is not merely correlated with neural activity, but also plays a pivotal role in stabilizing recent changes in synaptic efficacy. As such, localized disruption of IEGs Arc or c-fos by intrahippocampal administration of antisense oligonucleotides or germline disruption of the IEGs c-fos, tissue plasminogen activator, or zif268 impairs consolidation of long-term memory formation, without affecting learning or short-term memory. Further investigation into the expression and function of IEGs using catFISH and antisense approaches will likely increase understanding of the molecular and cellular bases of information processing involving the hippocampus. Hippocampus 2002;12:86–104. © 2002 Wiley-Liss, Inc.

Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ Hybridization (catFISH)

This protocol describes a method (cellular compartment analysis of temporal activity by fluorescent in situ hybridization or catFISH) that uses fluorescent in situ hybridization to immediate-early gene RNAs and confocal microscopy to identify neuronal populations activated at two distinct times. The combination of cellular and temporal resolution makes catFISH a valuable tool for investigating the dynamic interactions of neuronal populations associated with different behaviors or cognitive challenges.