Daun02 Inactivation of Behaviorally Activated Fos‐Expressing Neuronal Ensembles

Abstract

Learned associations about salient experiences (e.g. drug exposure, stress) and their associated environmental stimuli are mediated by a minority of sparsely distributed, behaviorally activated neurons coined 'neuronal ensembles'. For many years, it was not known whether these neuronal ensembles played causal roles in mediating learned behaviors. However, in the last several years the 'Daun02 inactivation technique' in Fos-lacZ transgenic rats has proved very useful in establishing causal links between neuronal ensembles that express the activity-regulated protein 'Fos' and learned behaviors. Fosexpressing neurons in these rats also express the bacterial protein b-galactosidase (b-gal) in strongly activated neurons. When the prodrug Daun02 is injected into the brains of these rats 90 min after a behavior (e.g. drug-seeking) or cue exposure, then the Daun02 is converted into daunorubicin by b-gal, which selectively inactivates the Fos and b-galexpressing neurons that were activated 90 min before the Daun02 injection. This unit presents protocols for breeding the Fos-lacZ rats and conducting appropriate Daun02 inactivation experiments. Significance StatementLearned behaviors are mediated by specific patterns of sparsely distributed neurons, called 'neuronal ensembles', that are selectively activated during behavior and cue exposures. For many years, neuroscientists observed that neural activity of specific neurons correlated with specific behaviors or cue exposures. However, they could not determine whether these selectively activated neurons play 'causal roles' in the learned behaviors because tools to selectively silence only the behaviorally activated neurons without affecting the surrounding neurons were unavailable. The Daun02 inactivation technique described in this protocol selectively inactivates neurons that were activated strongly enough during recent behavior to express the proteins b-galactosidase (b-gal) in Fos-lacZ transgenic rats. Researchers use this tool to establish whether specific neuronal ensembles directly mediate learned behaviors.

Cortical and amygdalar neuronal ensembles in alcohol seeking, drinking and withdrawal

“…Since 2009, a number of methods have been developed for selective manipulation of these Fos-expressing ensembles (Cruz et al, 2015; Cruz et al, 2013; Koya et al, 2009; Koya et al, 2016; Mayford and Reijmers, 2015; Tonegawa et al, 2015). However, to date, only the Daun02 inactivation procedure with Fos-lacZ transgenic rats has been used to assess Fos-expressing ensembles in alcohol research (de Guglielmo et al, 2016; Pfarr et al, 2015).…”

Mechanistic Resolution Required to Mediate Operant Learned Behaviors: Insights from Neuronal Ensemble-Specific Inactivation

“…The earliest technology used to demonstrate a causal role for behaviorally activated Fos-expressing neuronal ensembles in a learned behavior was the Daun02 inactivation procedure using Fos-LacZ transgenic rats (Bossert et al, 2011; Cruz et al, 2013, 2014, 2015; Koya et al, 2016). Indeed, the Daun02 inactivation procedure has been used in the vast majority of studies that examined Fos-expressing ensembles in operant learned behaviors.…”

“…Daun02 is catalyzed by βgal into daunorubicin, a toxic compound that leads to early inactivation of calcium-dependent action potentials (Engeln et al, 2016), followed by apoptosis and cell death only in the strongly activated βgal-expressing neurons (Pfarr et al, 2015). After a couple of days, the behavioral effects of the targeted neural inactivation by Daun02 can be assessed under a drug free condition (Bossert et al, 2011; Fanous et al, 2012; Koya et al, 2016) to demonstrate causal roles for neuronal ensembles within multiple nodes of the corticostriatal circuit, including prefrontal cortex, striatum and amygdala (Bossert et al, 2011; Cruz et al, 2013, 2014, 2015; Pfarr et al, 2015; de Guglielmo et al, 2016; Funk et al, 2016; Koya et al, 2016; Suto et al, 2016; Caprioli et al, 2017). …”

Methamphetamine and Ovarian Steroid Responsive Cells in the Posteriodorsal Medial Amygdala are Required for Methamphetamine-enhanced Proceptive Behaviors

“…Recently, Bruce Hope and colleagues have pioneered a technique that allows for determining whether specific neuronal ensembles are required for a specific behavior1415. Using Sprague Dawley rats expressing a cFos-lacZ transgene, the ‘DAUN02 inactivation technique’ exploits the expression of β- galactosidase (β-gal) under the cFos promoter to remove neurons that are activated by a specific stimulus or state while the neighboring non-activated neurons remain intact.…”

“…Daunorubicin is toxic to the cells expressing β-gal resulting in apoptosis161718 and thus a cell-specific lesion. As β-gal expression is induced only in neurons with a strong activation of the cFos promoter and not in Fos negative or weakly activated neurons1419, this method is a powerful approach for testing whether activation of specific neuronal groups or ensembles are necessary for an associated behavior141519.…”