TRPC channels are not required for graded persistent activity in entorhinal cortex neurons

Abstract

Adaptive behavior requires the transient storage of information beyond the physical presence of external stimuli. This short-lasting form of memory involves sustained ("persistent") neuronal firing which may be generated by cell-autonomous biophysical properties of neurons or/and neural circuit dynamics. A number of studies from brain slices reports intrinsically generated persistent firing in cortical excitatory neurons following suprathreshold depolarization by intracellular current injection. In layer V (LV) neurons of the medial entorhinal cortex (mEC) persistent firing depends on the activation of cholinergic muscarinic receptors and is mediated by a calcium-activated nonselective cation current (I_CAN ). The molecular identity of this conductance remains, however, unknown. Recently, it has been suggested that the underlying ion channels belong to the canonical transient receptor potential (TRPC) channel family and include heterotetramers of TRPC1/5, TRPC1/4, and/or TRPC1/4/5 channels. While this suggestion was based on pharmacological experiments and on effects of TRP-interacting peptides, an unambiguous proof based on TRPC channel-depleted animals is pending. Here, we used two different lines of TRPC channel knockout mice, either lacking TRPC1-, TRPC4-, and TRPC5-containing channels or lacking all seven members of the TRPC family. We report unchanged persistent activity in mEC LV neurons in these animals, ruling out that muscarinic-dependent persistent activity depends on TRPC channels.

Keywords: TRPC1/4/5 KO mice; hepta-TRPC KO mice; muscarinic; persistent firing.

Figure 1.

CCh-induced persistent firing in TRPC1/4/5-triple-knockout mice. Responses of mEC LV neuron to depolarizing current steps of different duration (left). Current pulse of equivalent strength was not able to elicit persistent firing after slight membrane hyperpolarization (right). Recording was obtained during blockade of neurotransmission with CNQX (10 μM), APV (30 μM) and picrotoxin (100 μM).

Figure 2.

qPCR analysis of the Trpc subunits. (a) qPCR analysis of the Trpc subunits made by RNA isolated from the entorhinal cortex (EC) together with the perirhinal cortex (PER) and the postrhinal cortex (POR). Quantitative expression analysis was performed using high efficiency probe-based assays. Data are given as mean ± SD (four animals). (b) qPCR analysis using SYBR green I assays specifically designed to verify the respective Trpc deletion in brain tissue of hepta-TRPC KO mice. The primer efficiency varied between assays and accordingly the level of expression is displayed as Cq values. Each bar represents the value of one animal.

Figure 3.

Muscarinic-dependent persistent activity in hepta-TRPC knockout mice. (a) CCh-induced persistent firing in mEC LV neuron in hepta-TRPC KO mice (left) and its complete block by the muscarinic antagonist atropine (1 μM; right). (b) Activity-dependence of persistent firing in hepta-TRPC KO mice. Responses of mEC LV neuron to depolarizing current steps of different duration. (c) Voltage-dependence of persistent firing in hepta-TRPC KO mice. Responses to depolarizing current steps at two differennt membrane potentials. The arrowhead indicates d.c. shift. (d) Box plots of firing frequency during 1 s current-step injection (initiation firing) and during persistent activity. No significant differences between WT control and hepta-TRPC KO mice were observed. (e) Frequency of persistent figing correlated positively with frequency of spike trains during current-step injections for WT control (black) and KO mice (red). Ploted data from d. Dotted lines represent the 95% confidence intervals. (f) Box plots of membrane depolarisation during persistent firing for WT control and KO mice. Box plots indicating median, 25th and 75th percentiles and individual vaslues. Whiskers show 5th and 95th percentiles, squares indicate mean. ns, not significant, t-test. All recordings were obtained during blockade of neurotransmission with CNQX, APV and picrotoxin.

Figure 4.

Induction and termination of persistent firing in hepta-TRPC knockout mice. (a) An example of a neurons that showed voltage-dependent persistent firing in response a brief stimulus (0.2 s, +0.4 nA). The arrowhead indicates d.c. shift. (b) Persistent firing termination by prolonged hyperpolarizing current pulse (6 s, −0.6 nA). All recordings were performed in the presence of CCh, CNQX, APV and picrotoxin.

Figure 5.

Graded persistent activity in hepta-TRPC knockout mice. (a) Repetitive stimulation with a 1 s depolarizing current step gives rise to four distinct increases of stable discharge rate in mEC LV neuron in hepta-TRPC KO mice. (b) Repetitive application of 6 s hyperpolarizing steps gives rise to discrete decreases of firing rate. Short intervals of firing are shown at an expanded time scale for each level below voltage traces. The lower diagrams correspond to the peristimulus histograms (bin width in (a) and (b): 300 ms and 700 ms respectively). All recordings were performed in the presence of CCh, CNQX, APV and picrotoxin.

Figure 6.

Activtiy dependent modulation of graded persistent firing in hepta-TRPC knockout mice. (a) Specimen trace showing that repetitive depolarizing current step gives initially increases of stable discharge rate followed, however, by its discrete decreases and full termination. The lower diagram correspond to the peristimulus histograms (bin width 700 ms). (b) Repetitive stimulation in same neuron after adding atropine. Recording was performed in the presence of CCh, CNQX, APV and picrotoxin.