These strategies will be useful both for characterizing the roles of the targeted genes and proteins as well as for manipulating the functions of the TRAPed population. The efficiency of Cre recombination is an important consideration for such experiments, given that we have found efficient Cre-dependent transgenes to be critical for successful TRAPing (data not shown). Fortunately, many high-efficiency transgenes identical in locus and design to the AI14 transgene used here have been developed

for Cre-dependent expression of fluorescent proteins, optogenetic learn more tools, and calcium indicators ( Madisen et al., 2012; Madisen et al., 2010; Zariwala et al., 2012). In addition, advances in site-specific transgenesis techniques now allow the rapid development of additional high-efficiency Cre-dependent transgenes ( Tasic et al., 2011). We have also successfully used TRAP in conjunction with viral expression of effector genes (data not shown). An understanding of the features of neuronal activity that lead to IEG expression and TRAPing will be important for applying TRAP. The relationship between synaptic activity and IEG expression

is not completely understood and appears to be dependent on many factors. In some cases, Y 27632 spiking alone is sufficient for IEG induction (Schoenenberger et al., 2009), whereas, in other cases, synaptic activation is critical (Luckman et al., 1994). The precise pattern of activity, as well as the duration and intensity of activity, affects IEG induction, and different IEGs have different thresholds of induction (Sheng et al., 1993; Worley et al., 1993). In addition, TRAP is binary (cells are either TRAPed or not), whereas IEG expression is graded (Schoenenberger et al., 2009; Worley et al., 1993). The probability of TRAPing is an unknown function of CreERT2 expression level during the critical time window surrounding TM or 4-OHT Linifanib (ABT-869) injection. Given that the functions relating recombination probability, IEG and CreERT2 expression level, and neuronal activity in TRAP are unknown,

the electrophysiological responses of the TRAPed population to the experimental stimulus are difficult to predict a priori. On one extreme, the TRAPed population may be a small, stochastic subset of a large population of cells that was weakly activated by the stimulus. On the other extreme, the TRAPed population may be a large percentage of a small population of cells that was strongly activated by the stimulus. Although more effort is necessary to fully distinguish between these possibilities, our observation of good correspondence between TRAPing and Fos expression in the cochlear nucleus (Figure 5) suggests that, at least in this system, the TRAPed population consists mostly of neurons that reliably express Fos at high levels in response to repeated presentation of the same stimulus.