Evidence that a defined population of neurons in lateral amygdala is directly involved in auditory fear learning and memory.

Memory is thought to be encoded within networks of neurons within the brain, but the identity of the neurons involved and circuits they form have not been described for any memory. Previously, we used fos-tau-lacZ (FTL) transgenic mice to identify discrete populations of neurons in different regions of the brain which were specifically activated following fear conditioning. This suggested that these populations of neurons form nodes in a network that encodes fear memory. In particular, one population of learning activated neurons was found within a discrete region of the lateral amygdala (LA), a key nucleus required for fear conditioning. In order to provide evidence that this population is directly involved in fear conditioning, we have analysed the expression of a key molecular requirement for fear conditioning in LA, phosphorylated Extracellular Signal Regulated Kinase 1 and 2 (pERK1/2). The only neurons in LA that specifically expressed pERK1/2 following auditory fear conditioning were in the ventrolateral nucleus of the LA (LAvl), in the same discrete region where we found learning specific FTL+ neurons. Double labelling experiments in FTL mice showed that a substantial proportion of the learning activated neurons expressed both pERK1/2 and FTL. These experiments provide clear evidence that the learning specific neurons we identified within LAvl are directly involved in auditory fear conditioning. In addition, learning specific expression of pERK1/2 was found in a dense network of dendrites contained within the border region of the LAvl. This network of dendrites may represent an activated dendritic field involved in fear conditioning in LA.

Tracking the fear memory engram: discrete populations of neurons within amygdala, hypothalamus, and lateral septum are specifically activated by auditory fear conditioning.

Memory formation is thought to occur via enhanced synaptic connectivity between populations of neurons in the brain. However, it has been difficult to localize and identify the neurons that are directly involved in the formation of any specific memory. We have previously used fos-tau-lacZ (FTL) transgenic mice to identify discrete populations of neurons in amygdala and hypothalamus, which were specifically activated by fear conditioning to a context. Here we have examined neuronal activation due to fear conditioning to a more specific auditory cue. Discrete populations of learning-specific neurons were identified in only a small number of locations in the brain, including those previously found to be activated in amygdala and hypothalamus by context fear conditioning. These populations, each containing only a relatively small number of neurons, may be directly involved in fear learning and memory.

The bile acid receptor TGR5 activates the TRPA1 channel to induce itch in mice.

BACKGROUND & AIMS: Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice. METHODS: Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice. RESULTS: TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gßγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice. CONCLUSIONS: BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

Identification of neurons specifically activated after recall of context fear conditioning.

The learning of new information and recall of that information presumably involves modification of and access to shared circuitry in the brain. However, learning and recall may involve the activation of distinct parts of that circuitry, according to the quite distinct functional differences between these two processes. Previously we examined neuronal activation following learning of context fear conditioning. Using the Fos-Tau-LacZ (FTL) transgenic mouse to label activated neurons, we identified a number of distinct populations of neurons in amygdala and hypothalamus which showed learning specific activation. These populations of neurons showed much less activation following recall. Here we ask what populations of neurons might be specifically activated following recall. We trained mice in context fear conditioning, and then looked at FTL activation following recall of context fear. We identified a number of populations of neurons which showed recall specific activation in nucleus accumbens shell, the anterio-medial bed nucleus of stria terminalis, the anterior commissural nucleus and the periventricular hypothalamic nucleus. These were all different populations of neurons compared with those activated following context fear learning. These different functional activation patterns occurring between learning and recall may reflect the different brain functions occurring between these two memory related processes.

Context fear learning specifically activates distinct populations of neurons in amygdala and hypothalamus.

The identity and distribution of neurons that are involved in any learning or memory event is not known. In previous studies, we identified a discrete population of neurons in the lateral amygdala that show learning-specific activation of a c-fos-regulated transgene following context fear conditioning. Here, we have extended these studies to look throughout the amygdala for learning-specific activation. We identified two further neuronal populations, in the amygdalo-striatal transition area and medial amygdala, that show learning-specific activation. We also identified a population of hypothalamic neurons that show strong learning-specific activation. In addition, we asked whether these neurons are activated following recall of fear-conditioning memory. None of the populations of neurons we identified showed significant memory-recall-related activation. These findings suggest that a series of discrete populations of neurons are involved in fear learning in amygdala and hypothalamus. The lack of reactivation during memory recall suggests that these neurons either do not undergo substantial change following recall, or that c-fos is not involved in any such activation and change.

Audiogenic seizure proneness requires the contribution of two susceptibility loci in mice.

Juvenile mice of the DBA/2J strain undergo generalised seizures when exposed to a high-intensity auditory stimulus. Genetic analysis identified three different loci underlying this audiogenic seizure proneness (ASP)-Asp1, Asp2 and Asp3 on chromosomes 12, 4 and 7, respectively. Asp1 is thought to have the strongest influence, and mice with only Asp1 derived from the DBA/2J strain are reported to exhibit ASP. The aim of this study was to characterise more accurately the contributions of the Asp1 and Asp3 loci in ASP using congenic strains. Each congenic strain contains a DBA/2J-derived interval encompassing either Asp1 or Asp3 on a C57BL/6J genetic background. A double congenic C57BL/6J strain containing both Asp loci derived from DBA/2J was also generated. Here, we report that DBA/2J alleles at both of these Asp loci are required to confer ASP because congenic C57BL/6 mice harbouring DBA/2J alleles at only Asp1 or Asp3 do not exhibit ASP, whereas DBA/2J alleles at both loci resulted in increased susceptibility for audiogenic seizure in double congenic C57BL/6 mice.

Congenic mouse strains enable discrimination of genetic determinants contributing to fear and fear memory.

The ability to learn and remember is variable within a population of a given species, including humans. This is due in part to genetic variation between individuals. However, only few genes have been identified that contribute to variation in learning and memory. Two inbred mouse strains, C57Bl/6J (B6) and DBA/2J (D2), show significant variation both in fear conditioning memory as well as primary responsiveness to fear. Several studies have identified quantitative trait loci (QTL) on chromosomes (Chr) 1 and 12 associated with performance in fear conditioning, but it is unclear if these QTL were associated with fear memory or innate fear responsiveness. To determine if these QTL are associated with fear memory or fear responsiveness, we studied congenic mouse strains harbouring D2-derived DNA from Chr1 or Chr12 on a B6 genetic background. Cohorts of D2, B6 and the congenic mice were tested throughout the process of fear conditioning by measuring a series of fear-related parameters. The Chr1 congenic mice showed clear deficits in fear memory compared to B6 mice, which established the presence of a QTL on Chr1 directly influencing fear memory. The Chr12 congenic mice also showed alterations in fear conditioning, but this was more associated with alterations in fear responsiveness. These findings thus provide evidence for the localisation of independent genetic determinants for fear memory and fear responsiveness.

A discrete population of neurons in the lateral amygdala is specifically activated by contextual fear conditioning.

There is no clear identification of the neurons involved in fear conditioning in the amygdala. To search for these neurons, we have used a genetic approach, the fos-tau-lacZ (FTL) mouse, to map functionally activated expression in neurons following contextual fear conditioning. We have identified a discrete population of neurons in the lateral amygdala that are activated specifically following learning. These neurons have the morphology of principal neurons of the amygdala, and are immunoreactive for glutamate. The highly specific localization of these neurons within the lateral amygdala suggests that these neurons may be a discrete population of neurons involved in fear learning.

A single exposure to an enriched environment stimulates the activation of discrete neuronal populations in the brain of the fos-tau-lacZ mouse.

Storage of experience, including learning and memory, is thought to involve plasticity within pre-existing brain circuits. One model for looking at experience-dependent changes is environmental enrichment (EE), which involves exposing animals to a complex novel environment. Animals exposed to EE have previously been shown to exhibit a variety of behavioural and structural alterations in the brain, including decreased stress, improved learning and memory, altered levels of immediate early genes and synaptic change in the visual cortex. We were interested in understanding what regions of the brain are activated during the initial stages of EE. We used fos-tau-lacZ (FTL) transgenic mice to examine changes in functional activation throughout the brain after a single exposure to EE. We found that there was a significant increase in FTL expression within particular morphologically identified neurons in a series of brain regions in the enriched group compared to control groups, indicating that multiple circuits were activated. These regions include the claustrum, infralimbic cortex, hippocampus, amygdala and the hypothalamus. The data suggest that EE stimulates an initial strong increase in activation of multiple functional circuits. These circuits are presumably involved in the initial response of the animal to the enriched environment.

A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections.

We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of beta-galactosidase (betagal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of betagal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.

Reduction of p75 neurotrophin receptor ameliorates the cognitive deficits in a model of Alzheimer's disease.

Alzheimer's disease (AD) is an extremely prevalent cause of dementia. It is characterized by progressive memory loss, confusion, and other behavioral and physiological problems. The amyloid-ß (Aß) protein is thought to be involved in the pathogenesis of AD, and there is evidence that Aß may act through the p75 neurotrophin receptor (p75) to mediate its pathogenic effects. This raises the possibility that reducing levels of p75 could be a treatment for AD by preventing the effects of Aß. In this study, we have crossed the transgenic AD model mice, Tg2576, with p75(-/-) mice to generate Tg2576/p75(+/-) mice with reduced levels of p75. These mice are rescued from the deficits in learning and memory and hippocampal function which were found in the Tg2576 mice. These findings suggest that reduction of p75 can ameliorate some of the primary symptoms of AD.

Tracing functional circuits using c-Fos regulated expression of marker genes targeted to neuronal projections.

We have developed novel techniques to trace functionally activated circuits and synaptic plasticity within the brain. We have generated transgenic mice, FTL, which contain a tau-lacZ fusion gene regulated by the promoter for c-fos. Following a particular nervous system stimulation in these mice, only neurons, which are functionally activated, will express LacZ, which is targeted to neuronal processes by the tau protein. In the FTL mice, we found highly inducible expression of lacZ by a range of different stimuli, and successful targeting of expression to neuronal cell bodies, axons and dendrites. To test if a functionally activated circuit could be visualized, the mice were deprived of water, which activates nuclei involved in body fluid homeostasis. LacZ was induced in these nuclei and their projections, allowing the mapping of a neuroendocrine circuit. Further studies have employed these mice in the analysis of neurons and circuits activated in vision, and learning and memory. We have also developed methods to measure markers of synaptic plasticity in the brain, and found significant experience dependent changes in the levels of these markers in different parts of the brain. We believe these techniques will aid in the identification of circuits for many different brain functions, and within those circuits, the locations of synaptic plasticity.

Neural crest cell lineage segregation in the mouse neural tube.

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.