Role of TRPC channels in temporal association tasks

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1. THEORETICAL BACKGROUND 
 
 
1.1. Introduction: From neuron to temporal association task 
During the last decades, memory has gained even more interest in the 
neuroscientific field thanks to new instruments and techniques that allow us to 
explore the brain in depth. Even with various differences among species, it is an 
important cognitive function present in mammals.  
Most of the memory functions, like encoding and retrieval are known to be 
especially important (Squire & Zola-Morgan, 1991). In particular, this work will 
focus on the ability of these circuits in associating events separated in time, 
known as “temporal associative learning” (Kitamura, Macdonald & Tonegawa, 
2015), trying to answer the following question: what makes the hippocampal area 
(Fig. 1) able to support the performance of 
such tasks?  
In the present chapter, the most recent 
theories about how the hippocampal area 
allows temporal associative learning will be 
explained, starting from single neuron 
channel properties. 
  
Dorsal 
Hippocampus 
Ventral 
Hippocampus 
Fig. 1. Dorsal hippocampus 
(blue) and ventral hippocampus 
(red) in mouse brain. Modiefied 
from Bannerman et al. (2014).
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1.2. TRPC Channels  
Transient Receptor Potential-Canonical (TRPC) channels are part of the super-
family of Transient Receptor Potential (TRP) Channels. TRPC channels in 
particular, aggregate a family of seven channels which are believed to work as 
store-operated as well as second messenger-operated channels in various cell 
types (Vazquez, Wedel, Aziz, Trebak & Putney, 2004). The TRPC family can be 
further divided into four subgroups according to their structural and functional 
similarities: TRPC1; TRPC2; TRPC3 - 6 – 7 and TRPC4 - 5. We focused on TRPC 
4 and 5 channels, which have similar structure (Montell, 2005) and are broadly 
expressed in the murine brain. 
While TRPC 1 (Fig. 2) is widely and generally expressed in mammals, 
TRPC 4 and 5 are enriched in the nervous system (Venkatachalam & Montell, 
2007). TRPC4 and 5 channels, are reported as 
the predominant TRPC subtypes in the adult 
rat brain, especially in the hippocampus 
(Fowler, Sidiropoulou, Ozkan, Phillips & 
Cooper, 2007). TRPC channels play a role as 
non-selective permeable Calcium cation (Ca
2+
) 
channels, which mainly control intracellular 
Ca
2+ 
concentration (Gees, Colsoul & Nilius 
2010).  
  
Fig. 2. Representation of a 
TRPC channel structure 
and their functional group. 
Modified from Montell 
(2005).
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1.3. TRPC channels and persistent firing 
Persistent neural activity also known as persistent firing (PF) refers to a special 
pattern of repetitive spiking activity in neurons that persist even when the trigger 
stimulus is over (Major and Tank 2004). Typically, this activity pattern ranges from 
hundreds of milliseconds to tens of seconds or even minutes (Reboreda, 2011). 
 Evidence of persistent firing has been found in several cerebral area such 
as the basal ganglia (Schultz, Tremblay & Hollerman, 2003), thalamus (Komura 
et al., 2001), superior colliculus (Kojima, Matsumura, Togawa & Hikosaka, 1996), 
brainstem (Moschovakis, 1997) , spinal cord (Prut & Fetz, 1999) and especially 
in many areas of cerebral cortex while performing working memory behaviours 
tasks requiring short-term retention of a sensory stimulus, like delayed match 
tasks (Fuster, 1995). The distribution of persistent activity in such a different set 
of brain areas, makes it a good candidate as very general and fundamental form 
of brain dynamics (Major & Tank 2004). 
 
1.3.1. Persistent firing in vitro 
Many experiments involving persistent firing have been carried out during the last 
twenty years. Research in vitro, using patch clamp recording techniques has 
given further insight into this special pattern of activity. Acetylcholine agonists like 
carbachol (Cch) allow persistent firing (Navaroli, Zhao, Boguszewski & Brown, 
2012), while muscarinic antagonists, like atropine, block persistent activity. 
Therefore, the start and the maintenance of persistent firing is muscarinic-
receptor dependent (Fig. 3).
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1.3.2. Persistent firing in vivo 
In addition to in vitro research, it is possible to study persistent firing in vivo. While 
in vitro experiments show persistent neural activity after a depolarizing pulse or 
synaptic stimulation in presence of a neuromodulator (Zhang & Seguela, 2010), 
when no external triggers are present, persistent firing must be explained by the 
internal dynamics of the cell or the circuit.  
Most of the explanations proposed rely on the important participation of 
non-specific Ca
2+
 sensitive cationic current (CAN Current) which as the name 
suggests, needs the presence of Calcium
1
. Activity-dependent stimulation of the 
CAN current can trigger a sustained depolarization, from the initial voltage level 
                                                           
1
 The Ca2+ dependency of the CAN current has been studied using different approaches. Strong buffering 
of intracellular Ca2+ close to 0 does not allow the “plateau” to be generated. Those experiments, together 
with the effect of the depletion of intracellular stores, point towards a different Ca2+ source to explain the 
Ca2+ dependency of the “plateau”. In this way, application of the L-type Ca2+ channel blocker nifedipine, 
Cd2], or Co2+, reduced the “plateau” and the CAN current (Reboreda, 2011) 
Fig. 3. Example of persistent firing in 
vitro slice preparation. (A) In control 
conditions, a neuron responds to 
current injection by spiking during 
current injection and then stopping 
when current injection ends. (B) After 
activation of acetylcholine receptors, 
the neuron spikes during current 
injection and then after the end of the 
current injection shows persistent 
spiking that continues for an 
extended period of minutes. (C) 
Example of self-terminating 
persistent firing in Cch in a different 
cell from above. Bottom trace shows 
timing of 2 s current injection for (a) - 
(c). (Modified from Yoshida & 
Hasselmo, 2009).
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(called “plateau”), that can outlast the stimulus for even several minutes. If this 
sustained depolarization is strong enough to reach the firing threshold, it could 
generate persistent firing (Reboreda, 2011). 
 This evidence makes TRPC channels, also due to their wide expression 
in cortical areas as mentioned before, excellent candidates to underlie CAN 
current and therefore persistent firing. Moreover, persistent neural activity has 
been recorded in areas rich in specifically TRPC 4 and TRPC 5 channels (Fowler 
et al., 2007; von Bohlen, Halbach, Hinz, Unsicker & Egorov, 2005). 
 
1.4. Role of Acetylcholine (ACh) in memory through persistent firing 
Several studies indicate that both muscarinic and nicotinic acetylcholine 
receptors play a role in encoding of new memories. Acetylcholine is supposed to 
enhance encoding by acting in many different ways: either increasing the strength 
of feedforward afferent input, while decreasing excitatory feedback activity 
mediating retrieval; by increasing theta 
rhythm oscillations and modulating 
hippocampal interneurons; by increasing 
the modification of synapses via Long Term 
Potentiation (LTP) and by activating 
intrinsic mechanisms for persistent spiking; 
(Hasselmo & Stern, 2006).  
We are mostly interested in how Ach can trigger intrinsic mechanism allowing 
persistent neural response.  
Fig. 4. Intrinsic mechanisms 
of regenerative spiking. 
(Modified from Hasselmo and 
Stern, 2006)
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A combination of extracellular ACh and spiking activity induced by current 
injection or synaptic stimulation activate the aforementioned CAN current, which 
increases Na+ influx and causes even more depolarization. This depolarization 
causes further spiking activity that causes calcium influx that further activates the 
CAN current, completing a cycle which could underlie persistent spiking (Blair, 
Kaczmarek & Clapham, 2009; Hasselmo & Stern, 2006; Major and Tank, 2004) 
(Fig. 4).  
This hypothesis is based on the intrinsic mechanism of repetitive activity 
generation, however, the first and oldest hypothesis about this pattern, relies on 
circuit connectivity and describes mnemonic activity as sustained by synaptic 
reverberation in a recurrent circuit. The idea claims that recurrent excitatory loops 
within a neural network can sustain a persistent activity in the absence of external 
inputs. 
Recently, experimental and modelling work has begun to test the 
reverberation hypothesis at the cellular level, using mostly the abundant 
excitatory recurrent connection in the CA3 area of the hippocampus (Kesner & 
Rolls, 2015) to explain “reverberating activity”. Nevertheless, it was recognized 
that reverberatory networks tend to be dynamically unstable, and possible 
hypotheses formed to understand whether stability can be reached according to 
this model, have still to be tested (Xiao-Jing Wang, 2001). Moreover, Jochems 
and Yoshida (2013) reported persistent firing in hippocampal CA3 pyramidal 
cells, supported by an intrinsic cellular mechanism. It has also been shown that 
elevated acetylcholine levels suppress CA3 recurrent connections (Kremin & 
Hasselmo, 2007; Vogt & Regehr, 2001).