Correlated basal expression of immediate early gene egr1 and tyrosine hydroxylase in zebrafish brain and downregulation in olfactory bulb after transitory olfactory deprivation☆
Highlights
► First basal expression study of immediate early gene egr1 in the entire zebrafish brain from larval into adult stages. ► Identification of cellular co-localization sites of egr1 with tyrosine hydroxylase (dopamine) in zebrafish brain. ► New double-fluorescence in situ hybridization/immunohistochemical protocol for confocal microscopy of zebrafish tissue. ► Unilateral olfactory deprivation experiments show downregulation of TH/egr1 expressing cells in zebrafish olfactory bulb.
Introduction
Immediate early genes (IEGs), such as the zinc-finger gene egr1 (one of the four early growth response family genes; synonyms: Zif268/NGFI-A/Krox-24/TIS8/ZENK) or the leucine zipper gene c-fos code for transcription factors which have been implicated in neural plasticity during neuronal activation via sensory or more complex forms of stimulation (Knapska and Kaczmarek, 2004, Poirier et al., 2008). Typically, egr1 upregulation occurs at higher integrative brain levels involved with learning and memory, in particular the mammalian hippocampus (Davis et al., 2003, Jones et al., 2001, Poirier et al., 2008). However, also extrahippocampal pallial areas, for example in songbirds and parrots (Jarvis and Mello, 2000, Jarvis and Nottebohm, 1997, Liedvogel et al., 2007, Mello et al., 1992, Mello et al., 1995, Mello and Clayton, 1994, Mouritsen et al., 2005), or the pallium and acoustic midbrain of female túngara frogs (Burmeister et al., 2008, Mangiamele and Burmeister, 2008) may be involved.
The egr1 gene has been linked to neural activation also in teleosts. Generally, egr1 upregulation is present in the teleostean brain as a response to systemic kainic acid stimulation which activates glutamatergic receptors (shown in a cichlid species; Burmeister and Fernald, 2005). A more specific case has recently been reported for electrosensory South-American knifefishes (Apteronotus leptorhynchus; Harvey-Girard et al., 2010). These weakly electric fish emit wave-type electric organ discharges and sense individual frequency differences of conspecifics. They respond with so-called chirps (transient change of frequency) to a new individual frequency in the environment until they adapt to its presence and, thus, to that of its emitter. Correlated with this learning process is a transient upregulation of egr1 in pallial (presumably the hippocampal homologue and additional) areas. Thus, depending on the species-specific behaviors, egr1 is upregulated during sensory activation and/or long-term memory formation in various pallial divisions of the vertebrate telencephalon.
Imprinting on kin versus non-kin has recently been shown to occur in zebrafish during a 24 h time window at day six of larval development and to depend on olfactory cues (Gerlach et al., 2008, Mann et al., 2003). During larval life, this allows formation of shoals based on kin association (Gerlach et al., 2007). In adult zebrafish, kin recognition is important for inbreeding avoidance (Gerlach and Lysiak, 2006). Therefore, it is possible that immediate early genes such as egr1 are involved in the process of imprinting itself, but also in subsequent kin recognition, a form of learned long-term memory. We therefore sought to investigate the possible role of egr1 in olfactory processing and learning. Two areas are of immediate interest in this context: the olfactory bulb and the medial amygdala.
The peripherally lying glomeruli in the olfactory bulb (OB) of all vertebrates are recipient of olfactory epithelial sensory cell axons which synapse on dendrites of deeper lying efferent mitral and tufted cells (review in Cave and Baker, 2009). In mammals, periglomerular cells modulate this glutamatergic synaptic transmission both by releasing gamma-aminobutyric acid (GABA) and dopamine (DA). In contrast, the centrally lying small granule cells of the OB modulate mitral cell activity at the latter's secondary dendrites only via GABA. This modulatory network underlies plastic changes depending on olfactory experience (Mandairon and Linster, 2009). For example, although egr1 has a strong basal expression in mammalian OB inner granule layer and a subset of periglomerular OB cells, egr1 upregulation in periglomerular cells differs locally depending on the odorant (Inaki et al., 2002) and glomerular layer activity levels are dependent on olfactory experience (Woo et al., 2007). Furthermore, odor deprivation through naris occlusion and other forms of olfactory epithelium deprivation experiments in mice and rats show that tyrosine hydroxylase (TH; marker for DA cells) is downregulated in periglomerular cells, whereas glutamic acid decarboxylase (GAD, marker for GABA cells) in these (and inner granule) cells is not downregulated (Baker et al., 1983, Baker et al., 1993). Subsequent naris occlusion experiments revealed a correlated downregulation of egr1 and TH expression levels in a subset of dopaminergic periglomerular cells, but no egr1 downregulation in inner granule cells (Akiba et al., 2009). Thus, egr1 appears to mediate activity-dependent TH-expression in OB dopaminergic periglomerular neurons. Moreover, strong induction of egr1 expression is seen in rodent olfactory bulb periglomerular and inner granule cells in response to odor enrichment (Mandairon et al., 2008) and in inner granule cells in response to novel – but not to familiar – olfactory stimuli (Busto et al., 2009).
As mentioned above, the initial imprinting process in 6 days postfertilization (dpf) zebrafish larvae allows older larvae and adult fish (for example females) to perceive these learned olfactory kin signals. This olfactory kin signal activation might possibly be reflected in the pattern of egr1 and TH expression in the OB. The zebrafish OB principally shows the same layers and cell types as its mammalian counterpart (Byrd and Brunjes, 1995). However, the zebrafish efferent mitral cell bodies do not form a distinct layer between peripheral glomerular and inner granule cell layers, as they may also be located in the glomerular layer. In addition to mitral cells, also ruffed cells (described by Fuller and Byrd, 2005) project to the zebrafish brain (Fuller et al., 2006, Rink and Wullimann, 2004). Teleostean ruffed cells may thus correspond to mammalian tufted cells, a second efferent OB population in mammals. TH positive periglomerular cells are also present in the zebrafish glomerular layer (Fuller et al., 2006, Yamamoto et al., 2011). Deprivation experiments involving the detergent Triton X-100 induce a transient loss of olfactory sensing (Friedrich and Korsching, 1997) and recovery of olfactory sensory cells after 2–5 days (Iqbal and Byrd-Jacobs, 2010). Furthermore, loss of TH expressing cells after permanent deafferentiation has also been reported (Byrd, 2000). However, the relationship of TH expression to that of egr1 has not been looked at since the expression of egr1 has only been reported for the embryonic zebrafish where the OB is negative for egr1 (Close et al., 2002).
Another site of co-expression of egr1 and TH is seen in the mammalian medial amygdala. For example in male prairie voles, egr1 expression is modulated by social contact in medial amygdalar dopamine cells (Northcutt and Lonstein, 2009). This part of the vertebrate amygdala is recipient of vomeronasal information, often involved with processing of pheromones (Martínez-García et al., 2009). Although teleosts do not have a separate vomeronasal organ in addition to the main olfactory epithelium (Eisthen, 1997, Eisthen, 2004) they do have sensory neurons carrying the corresponding class of receptors (Hansen et al., 2003, Hansen et al., 2004). Interestingly, dopamine cells do occur in the teleostean telencephalic area called the supracommissural nucleus of area ventralis (Vs) which has been hypothesized to represent part of the subpallial amygdala (Mueller et al., 2008, Northcutt, 2006, Northcutt and Braford, 1980, Wullimann and Mueller, 2004). The Vs, thus, possibly contains the teleostean medial amygdala. This subpallial area is in receipt of secondary olfactory projections from the OB in teleosts (see for example Northcutt, 2006). In the zebrafish, Vs also receives extrabulbar projections from the olfactory epithelium (Gayoso et al., 2011), and additionally, mitral cells of a specialized dorsomedial glomerular field in the olfactory bulb receiving input mostly from crypt cells project to Vs (Gayoso et al., 2012). Thus, the Vs potentially may be involved in imprinting related processes.
Therefore, in this basic paper we first established the developmental profile of basal egr1 expression by in situ hybridization (ISH) beyond the known embryonic expression (Close et al., 2002) in larval and adult zebrafish. We then performed double-labeling for TH (immunohistochemistry, IHC) and egr1 transcripts (ISH) in adult imprinted zebrafish in order to check for co-localization of both markers in the OB and additional brain centers of interest, such as the suspected medial amygdala (Vs). We then subjected adult to transient deafferentiation of olfactory input in order to see the effect on downregulation of these markers in the OB. Our results indicate that a similar pattern of basal egr1 brain expression exists throughout development (forebrain and alar plate midbrain) and that TH positive cells do co-localize with egr1 transcripts in periglomerular OB cells and various additional TH positive brain centers (for example Vs), but not in others. Furthermore, strong downregulation of both markers is seen after transient olfactory deprivation in the OB. This indicates similar roles of egr1/TH in olfactory processing in zebrafish and mammals.
Section snippets
Animals and rearing conditions
Wild type zebrafish from the Gerlach lab (Carl von Ossietzky University Oldenburg, Department of Biology and Environmental Sciences, Animal Biodiversity and Evolutionary Biology, Oldenburg, Germany) and from our own facility were used in this study. Zebrafish were kept and bred according to standard procedures (Westerfield, 1995). For breeding, each female was housed with one male in a 3 l tank. In the afternoon, egg dishes covered with a mesh were put into the tanks and examined the following
Expression profile of basal egr1 expression during zebrafish brain development
Our observations on egr1 larval brain expression show that this gene maintains its expression within the forebrain and alar plate mesencephalon beyond the previously described embryonic pattern of basal egr1 expression in the zebrafish brain (Close et al., 2002). We confirm essentially the reported embryonic domains (retina, telencephalon, hypothalamus, preoptic region, diencephalon, optic tectum), except for the (probably erroneously) reported embryonic midbrain tegmental domain, as we did not
egr1 has a high fore- and midbrain basal expression profile from the embryonic into the adult brain, but is almost absent from hindbrain
There is an impressive similarity of basal egr1 expression in the adult mouse (Christy et al., 1988, Schlingensiepen et al., 1991) and rat (Herdegen et al., 1990, Herdegen et al., 1993, Herdegen et al., 1995) review: (Beckmann and Wilce, 1997) brain compared to our findings in the zebrafish brain. Generally, high expression levels are found in all three species in the pallial and subpallial telencephalon, as well as in the diencephalon (pretectum, thalamus, hypothalamus) and (alar plate)
Acknowledgements
We would like to thank Bea Stiening for expert technical assistance, Dr. Olga Alexandrova for help with photography and Photoshop, and Prof. H.-.J Tsai (National Taiwan University) for the donation of a plasmid used to make probes for egr1 transcripts in this study. Gabriele Gerlach and Cornelia Hinz (Carl-von-Ossietzky Universität Oldenburg, Germany) helped unreservedly during animal retrieval and behavioral tests performed in Oldenburg, Rainer Friedrich and Iori Ito (Friedrich Miescher
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Grant sponsors: This work was funded by the Deutsche Forschungsgemeinschaft, Bonn, Germany (SPP Olfaction 1392, Project Wu211/2-1) and the Graduate School for Systemic Neurosciences at the LMU-Munich, Planegg, Germany.