Working memory is a type of memory that is active only for a short period of time (Fuster and Alexander, 1971; Goldman-Rakic, 1992). A common example of working memory is our ability to hold a phone number in our minds transiently, until it is dialed. Working memory is critical for many cognitive tasks, such as making decisions and guiding subsequent actions (Goldman-Rakic, 1992; Wickelgren, 2001). Deficits in working memory are associated with numerous pathological conditions, including schizophrenia, attention deficit hyperactivity disorder, aging, and stress (Birnbaum et al., 2004; Goldman-Rakic, 1992; Goldman-Rakic and Selemon, 1997). Therefore, it is important to understand the neural basis of working memory. During performance of a working memory task, pyramidal neurons in prefrontal cortex (PFC) are able to maintain sustained firing during a delay period between an informative cue and the appropriate behavioral response (Goldman-Rakic, 1995). Thus, stimulus-specific persistent neural activity is thought to be a neural substrate for holding memories over short time delays (Major and Tank, 2004). Once persistent activity is triggered within a neuron or neural circuit, its activity can be maintained after the stimulus has terminated. Three general (non-mutually exclusive) mechanisms of persistent activity have been hypothesized: recurrent network activity (Compte et al., 2000; Wang, 2001), short-term synaptic plasticity (Mongillo et al., 2008) and intrinsic biophysical cellular properties. Several studies have demonstrated the role of intrinsic biophysical cellular properties in persistent activity (Egorov et al., 2002; Egorov et al., 2006; Fransen et al., 2006). This firing behavior is linked to cholinergic muscarinic receptor activation and phospholipase C (PLC) signaling in the absence of synaptic reverberations. Two fundamental questions are: (1) What mechanism underlies the generation of sustained firing at a single cell level? (2) What role does intrinsic persistent firing play in working memory? Pharmacological studies suggest that persistent activity relies on activity of Ca2+-activated non-selective cation (CAN) current (ICAN) (Egorov et al., 2002; Egorov et al., 2006). However, the molecules that constitute CAN channels in the brain are not well studied, and the importance of CAN channels to working memory is unknown.;I seek to identify molecular mechanisms to convert subthreshold input into intrinsic persistent neural firing in PFC layer 5 pyramidal neurons. I hypothesize that CAN channels are responsible for the intrinsic properties that mediate persistent neural activity in PFC layer 5 neurons. During muscarinic receptor activation, bursts of action potentials will lead to Ca2+ influx. CAN channels will be activated due to the increased intercellular Ca2+ and promote a slow afterdepolarization (sADP), a transition state between subthreshold input and suprathreshold sustained firing. If the sADP is strong enough, it will trigger subsequent spikes, causing further opening of voltage-dependent Ca2+ channels and Ca2+ influx, and thus further opening of CAN channels. Therefore, ICAN will be maintained by a positive feedback loop, generating persistent activity. I have combined electrophysiology, pharmacology, genetics and behavioral analyses to address the potential roles of CAN channels and persistent activity in working memory.;First, I confirmed that in the presence of the muscarinic agonist carbachol a brief burst of action potentials triggers a prominent sADP and persistent activity in these neurons. Second, I confirmed that this sADP and persistent firing require activation of a PLC signaling cascade and intracellular calcium signaling. Third, I obtained direct evidence that the transient receptor potential melastatin 5 channel (TRPM5), which is thought to function as a CAN channel in non-neural cells, makes an important contribution to sADP and persistent activity in the layer 5 neurons. Importantly, Trpm5-/- mice show deficits in a Delayed-Non-Match-to-Sample maze (DNMTS) task, a working memory task in the mouse model. Furthermore, PFC-specific expression of TRPM5 using a virally-mediated delivery system in Trpm5-/- mice produced a partial rescue of deficits in the working memory tasks, indicating the importance of TRPM5 in mPFC for performance of these tasks. Lastly, I found that PFC-specific expression of TRPM5 partially rescued the electrophysiological defects in Trpm5-/- mice. By identifying an ion channel contributing to working memory, this work opens the possibility of discovering new drugs for treating working memory deficit.
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