# A protocol for inducing long-term plasticity of intrinsic neuronal excitability in neurons of the dorsal lateral geniculate nucleus

Michaël Russier, Maël Duménieu, Anushka Wakade, Salvatore Incontro, Laure Fronzaroli- Molinieres, Dominique Debanne

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## To cite this version:

Michaël Russier, Maël Duménieu, Anushka Wakade, Salvatore Incontro, Laure Fronzaroli- Molinieres, et al.. A protocol for inducing long-term plasticity of intrinsic neuronal excitability in neurons of the dorsal lateral geniculate nucleus. Journal of visualized experiments: JoVE, 2024, 20, pp.211. hal-04876338

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Distributed under a Creative Commons Attribution 4.0 International License
**TITLE:**

A protocol for inducing long-term plasticity of intrinsic neuronal excitability in neurons of the
dorsal lateral geniculate nucleus

AUTHORS AND AFFILIATIONS :

Michaël Russier*, Maël Duménieu, Anushka Wakade, Salvatore Incontro, Laure Fronzaroli-Molinieres & Dominique Debanne*

UNIS, UMR1072, INSERM, Aix-Marseille University, France

*Corresponding authors [michael.russier@univ-amu.fr](mailto:michael.russier@univ-amu.fr) or [dominique.debanne@univ-amu.fr](mailto:dominique.debanne@univ-amu.fr)

SUMMARY:

We describe here a protocol for inducing long-term plasticity of neuronal intrinsic excitability in relay neurons from the dorsal lateral geniculate nucleus maintained in *ex vivo* brain slices.

ABSTRACT:

The dorsal lateral geniculate nucleus (dLGN) has long been held to act as a basic relay for
visual information travelling from the retina to cortical areas, but recent findings suggest largely
underestimated functional plasticity of dLGN principal cells. However, the cellular mechanisms
supporting these changes have not been fully explored. We report here a protocol to induce
long-term potentiation of intrinsic neuronal excitability (LTP-IE) in dorsal dLGN relay cells from
acute brain slices of young rats. Intrinsic plasticity is generally induced in parallel with synaptic
plasticity. However, in dLGN neurons, LTP-IE is reliably induced by spiking activity at a
frequency of 40 Hz during 10 minutes. LTP-IE in dLGN relay neurons is long-lasting as it can be
followed up to 40 min after the induction protocol. In conclusion, our results provide the first
evidence for induction of intrinsic plasticity in dLGN relay cells, thus further pointing the role of thalamic neurons in activity-dependent visual plasticity.

## INTRODUCTION:

The overall goal of this method paper is to provide a simple way for inducing long-lasting plasticity of neuronal excitability in visual thalamic neurons of the rat *in vitro*, using standard current clamp mode of the patch clamp technique ¹, ². The rational behind the development of this technique is its simplicity and reproducibility. The advantages over alternative techniques such as stimulation of synaptic inputs paired or not with postsynaptic action potentials delivered with a given timing is its reliability.

Plasticity in the visual system is traditionally thought to be exclusively expressed at the cortical level whereas the dorsal lateral geniculate nucleus (dLGN), a primary recipient structure of retinal inputs at the thalamic level, is traditionally considered to be just a relay of visual information ³. However, this initial conclusion has been challenged by recent works indicating that this simplistic view does not hold longer ⁴⁻⁶. For instance, functional deficits in visual response are already observed in amblyopic patients at the stage of the LGN ⁷. In addition, a large proportion of dLGN relay neurons in a given monocular territory receive inputs from each eye, indicating a potential binocularity for large proportion of dLGN neurons ⁸⁻¹⁰. Moreover, monocular deprivation produces significant shift in ocular dominance in dLGN neurons ⁵, ¹¹, ¹².

Among the mechanisms of functional plasticity that may occur in ocular dominance shift, plasticity of intrinsic neuronal excitability is a potential candidate. Indeed, many brain regions including visual areas express intrinsic plasticity following various behavioural tasks ¹³, ¹⁴. Long-term intrinsic plasticity has been reported in central neurons following stimulation of afferent glutamate inputs ¹⁵⁻¹⁸, spiking activity ¹⁹, ²⁰, sensory stimulation ²¹ or following activity or sensory deprivation ²⁰, ²², ²³. We describe here a protocol allowing the induction of long-term potentiation
of intrinsic excitability (LTP-IE) in dLGN relay neurons following spiking activity at a frequency of 40 Hz. The 40 Hz firing frequency corresponds to a frequency observed in most dLGN neurons *in vivo* upon visual stimulation<sup>24, 25</sup>.

## PROTOCOL:

### 1. Animals

1.1. Experiments were conducted using 19 to 25 days old Long Evans rats of both sexes (weight ranging between 50 and 90 g).

1.2. Animals were housed in conventional plastic cages, together with their mother and their littermate. Temperature was kept constant with a 12 h light/dark cycle and ad libitum access to water and food.

### 2. Acute slices of rat dLGN

All experiments were conducted according to the European and Institutional guidelines (Council Directive 86/609/EEC and French National Research Council and approved by the local health authority (Veterinary services, Préfecture des Bouches-du-Rhône, Marseille)).

2.1. Prepare cutting/recovery and extracellular recording solutions. Cutting/recovery solution contains (in mM): 92 n-methyl-D-glutamine, 30 NaHCO₃, 25 D-glucose, 10 MgCl₂, 2.5 KCl, 0.5 CaCl₂, 1.2 NaH₂PO₄, 20 Hepes, 5 sodium ascorbate, and 3 sodium pyruvate and bubbled with 95% O₂ - 5% CO₂ (pH 7.4). Cutting solution must be kept cold during all the slicing procedure. Recording solution (artificial cerebrospinal fluid, ACSF) contains (in mM): 125 NaCl, 26 NaHCO₃, 2 CaCl₂, 2.5 KCl, 2 MgCl₂, 0.8 NaH₂PO₄, and 10 D-glucose and must permanently be equilibrated with 95% O₂ - 5% CO₂.
2.2. Prepare the dissecting tools and two ice platforms (Figure 1A).

2.3. Put ice in the outer tank and fill the slicing chamber of the vibratome with ice-cold cutting solution (Figure 1B).

2.4. Deeply anesthetize young Long Evans rat (age: P19-P25) with isoflurane (5%) and kill the animal by decapitation with either scissors or a guillotine (depending on the age/size of the animal) at the level of the medulla. Put the head on the first iced platform and water the isolated tissue every 10 s with the oxygenated ice-cold slicing solution.

2.5. Cut the scalp in caudal direction and using small scissors cut the skull bilaterally. Using blunt forceps, open the skull and rapidly extract the brain from the skull with a spatula and put it on the second ice platform. The time should not exceed 2 min from the decapitation to the extraction of the brain.

2.6. Make 2 cuts in the frontal plane of the brain over the entire brain; the first to remove the anterior part of the cortex and the olfactory bulbs (see **Figure 1C**) and the second at the level of the inferior colliculus to remove the posterior part of the cortex and the cerebellum.

2.7. Glue the brain block on the plate of the vibratome with the rostral side up (see **Figure 1D** & **1E**). Brain must be regularly watered (every 10 seconds) during the whole procedure until it is submerged in the slicing chamber of the Vibratome.

2.8. Make 350 µm thin slices containing the dLGN with the vibratome (Figure 1G). Remove the cortex and the hippocampus from the midbrain with a pencil by gently pulling on
these structures.

2.9. Aspire the slices with a broken Pasteur pipette connected to a flexible rubber sucker (Figure 1H).

2.10. Let the slices recovering during 20-30 minutes in the recovery solution (Figure 1I).

3. Whole-cell patch-clamp electrophysiology

For whole-cell patch-clamp recordings from dLGN relay neurons, use a specific patch-clamp
amplifier.

3.1. Turn on the pumps of the rig to make the extracellular recording saline (see composition in 2.1) circulating in the recording chamber.

3.2. Prepare the intracellular solution (in mM): 120 K-gluconate, 20 KCl, 10 Hepes, 0.5 EGTA, 2 MgCl₂, 2 Na₂ATP and 0.3 NaGTP (pH 7.4). Intracellular solution can be prepared in advance and kept frozen at -80°C. When unfrozen, the solution is kept at 4°C.

3.3. Add the GABA<sub>A</sub> channel blocker, picrotoxin (100 µM final concentration, AbCam) and the ionotropic glutamate receptor antagonist, kynurenate (2 mM, final concentration) to the extracellular saline.

3.4. Transfer the slice to a submerged chamber mounted on an upright microscope. The chamber is temperature-controlled at 30°C and continuously perfused with equilibrated aCSF.
3.5. Place the U-shaped platinum wire on the slice and position the slice to visualize the dLGN (Figure 2A).

3.6. Prepare patch pipettes from borosilicate glass tube pulled with a puller. The patch pipettes should have a resistance of 5-10 MΩ when filled with the intracellular solution.

3.7. Visualize the dLGN at high magnification (X40 or X60) and select a neuron for patch-clamp recording using differential interference contrast infrared video-microscopy (Figure 2B). The selection of neurons is made on their healthy appearance (i.e., slightly swollen with sharp membrane contours).

3.8. Using the micromanipulator, position the patch-pipette on the selected neuron with constant positive pressure. Set the amplifier on VC mode and inject a 5-mV voltage step. Set the voltage to -65 mV. Then, release the positive pressure to obtain a seal of high resistance (corresponding to the reduction of the current in response to the voltage steps). Aspire slightly through the pipette to obtain clear transients. Set the amplifier on CC mode, balance the bridge to compensate access resistance and hold the neuron at -65 mV.

## 4. Data acquisition

4.1. Acquire signals at 20 KHz to better detect the fast action potentials. Set the low pass filter to 10 kHz for voltage and current signals.

4.2. Monitor neuronal excitability during a control period of about 10 minutes by injecting positive pulse of current (typically ~80-250 pA during 500 ms) sufficient to elicit 3-5 action potentials in control conditions at a frequency of 0.1 Hz. The input resistance of
the neuron is monitored throughout the duration of the experiment with a brief negative
pulse of current (typically -20 pA during 100 ms).

NOTE: the amplitude of the current pulse must be kept constant before and after induction of LTP-IE.

5. Induction of LTP-IE

5.1. After the control period, induce LTP-IE by eliciting trains of 15 spikes evoked by 15 short steps (2-5 ms) of depolarizing current delivered at 40 Hz during 10 minutes. Choose the amplitude of the current pulse to elicit a single action potential each time (i.e., 15 action potentials per train). Each train of 15 spike is elicited at a frequency of 0.1 Hz (i.e., 60 trains and 900 spikes).

6. Data analysis

Analysis procedure is the same before (control conditions) and after LTP-IE induction.

6.1. For each recorded trace, calculate the input resistance (R_in) from voltage response to the small negative current pulse injected before the test step of current (R_in=V_step/I_step). If there is a run-up or a run-down of the input resistance (more than 10%) during the control period, the experiment must be discarded.

6.2. Select recorded traces based on the membrane potential before any current injection.
Keep only the traces with a resting membrane potential from -67 to -63 mV.

6.3. Quantify LTP-IE was quantified over a period of 10 minutes, 20 minutes after the beginning of the post-induction period by measuring the number of AP evoked by each current pulse and averaged each minute by using a home-made routine in Igor Pro (see Supplementary Information). In brief, spikes were detected when the voltage signal
crossed 0 mV. LTP-IE is expressed as the number of action potential elicited by the
current pulse injection after the induction protocol compared to before (%). The analysis
was made on -10-0 min for the baseline and +20/+30 min for the test.

REPRESENTATIVE RESULTS:

dLGN neurons were recorded in whole-cell configuration and LTP-IE was induced by action potential firing at 40 Hz during 10 min in the presence of ionotropic glutamate and GABA receptors antagonists (Figure 3A). A three-fold increase in the number of action potential was observed 20-30 minutes after the induction (Figure 3B) without any change in input resistance, R<sub>in</sub> (Figure 3C). This protocol reliably induced LTP-IE in dLGN neurons (Figure 3D) <sup>26</sup>. These results therefore demonstrate how the protocol described here, is efficient for inducing long-lasting plasticity of neuronal excitability in dLGN neurons *in vitro*.

FIGURE AND TABLE LEGENDS:

**Figure 1.** Method for obtaining brain slices containing the dLGN. A. Tools necessary for extracting the brain. B. Slicer. C. Cutting of the brain in frontal plans. D. Glue on the plate. E. Disposition of the brain on the plate. F. Brain glued on the plate. G. Slicing. H. Taking of the slice. J. Slices in the recovery solution.

**Figure 2.** View of the slice containing the dLGN and visualization of neurons under the microscope. A. Global view with the objective X4 of the slice containing the dLGN and the vLGN and immobilized in the recording chamber by a nylon thread. B. View with the objective x40 of dLGN neurons in the contralateral region of the dLGN symbolized by the dotted rectangle in panel A.
**Figure 3.** Induction of LTP-IE in a dLGN neuron. A, recording configuration and induction stimulus. Stimulus used for LTP-IE induction consisted of 15 pulses of ~1 nA to elicit an action potential by each depolarizing current pulse. B, time-course of LTP-IE. Top, representative traces before (black) and after (red) LTP-IE induction. Bottom, time-course of the normalized action potential number per depolarizing pulse as a function of time. Note the increased spike number that does not show any decline over >40 min. Adapted from Duménieu et al., BioRxiv 2023. C, stability of input resistance. Deflection of potential induced by a negative step of current before (black) and after (red) induction of LTP-IE. Input resistance is measured at fixed latency (symbolized by *). D, left, group data on 8 dLGN neurons showing the reproducibility of LTP-IE. Right, group data on 8 dLGN neurons showing the lack of R<sub>in</sub> change.

**DISCUSSION:**

We report here the induction of LTP-IE in dLGN neurons maintained alive in acute brain slices by stimulation of the recorded neuron to evoke action potentials at a frequency of 40 Hz for 10 min. This protocol is simple to implement in any neurophysiology lab as it requires a minimal number of equipment (slicer, microscope, 1 amplifier, 1 acquisition board and computer). However, a few critical steps must be respected in order to collect valuable data. The first critical step within the protocol is the quality of the brain tissue. A healthy thalamic slice contains many neurons from the surface to the middle of the slice (~150 µm). Healthy neurons appear slightly swollen whereas dying neurons are usually shriveled. The second critical step is the stability of the recording during the baseline. The number of action potentials should be stable without any run-up or run-down. In addition, the input resistance tested with a brief pulse of hyperpolarizing current should not display any change larger than 10%. Finally, the holding
current must be stable within +/- 10 pA. Such induction protocol has been first used to induce LTP-IE in visual cortical neurons <sup>19, 20</sup>. We show here that this protocol is also able to induce intrinsic potentiation in thalamic relay neurons of the visual system <sup>26</sup>. The induction of spikes with direct current injection is supposed to mimic the retinal input stimulation that normally drives dLGN neurons as stimulation of single retinal fiber usually evokes excitatory post-synaptic potential (EPSP) that is able to trigger an action potentials in dLGN neuron <sup>27, 28</sup>. The expression mechanisms of LTP-IE in dLGN neurons have been investigated in the original publication <sup>26</sup> and revealed that Kv1 channels are down-regulated following induction of LTP-IE. Thus, down-regulation of Kv1 channels is responsible for both LTP-IE in fast-spiking cortical interneurons <sup>16</sup> and in dLGN neurons <sup>26</sup> and for homeostatic plasticity in the CA3 region <sup>29, 30</sup>. However, the signaling pathway differ as PKA is involved in dLGN neurons, while a major role for mTOR has been suggested in fast-spiking interneurons.

The present protocol provides reproducible results in the experimental conditions used here : 3 mM Ca<sup>2+</sup>, 2 mM Mg<sup>2+</sup>, picrotoxin and kynurenate, 10 min stimulation at 40 Hz in rats aged 19-25 days <sup>26</sup>. Indeed, on average, this protocol allows a 2-fold increase in excitability. The magnitude of the enhanced excitability decreases with the duration of stimulation, frequency of stimulation or age of the animal <sup>26</sup>. Before eye opening, no LTP-IE can be observed. The frequency used to induce long-lasting plasticity of intrinsic neuronal excitability corresponds to the gamma band of brain oscillations that is known to be the binding frequency for sensory perception <sup>31</sup> and to promote plasticity in the visual system <sup>32</sup>.

However, if a lower calcium concentration was used (i.e., 1.5 mM Ca<sup>2+</sup>) the protocol probably needs to be revised as it has been previously shown for synaptic plasticity <sup>33</sup>. In this case, the frequency of stimulation will probably need to be elevated to 60-80 Hz. The incidence of GABA receptor blockers (picrotoxin of gabazine) used in the present protocol on the induction of LTP-IE is thought to be limited as spikes are evoked by direct current injection. Nevertheless, in the *in vivo* situation where action potentials are evoked in dLGN neurons by retinal
stimulation, recruited GABAergic input<sup>34</sup> would probably elevate spike threshold. Thus, stronger
retinal inputs would probably be necessary to trigger LTP-IE in dLGN neurons. However,
neuromodulator systems active during arousal such as orexin may reduce the threshold<sup>35</sup>.

Induction of long-lasting intrinsic plasticity in dLGN relay neurons can be achieved either
through visual deprivation, stimulation of retinal inputs to evoke action potentials or directly by
eliciting action potentials through current injection in the recorded neurons <sup>26</sup>. This last protocol
is by far the simplest method because it is fast to implement and it provides equivalent results to
the synaptic simulation procedure <sup>26</sup>.

This protocol has been shown to induce LTP-IE in both visual cortical pyramidal neurons
<sup>19</sup> and in thalamic relay neurons <sup>26</sup>. It could probably also be used for inducing long-lasting
plasticity of neuronal excitability in other subcortical visual nuclei including the ventral LGN
(vLGN) or the superior colliculus (SC).

## ACKNOWLEDGMENTS:

Supported by INSERM, CNRS (to DD), AMU (to MR), FRM (DVS20131228768 to DD and DEQ20180839583 to DD), NeuroSchool ("France 2030" program via A*Midex (Initiative d'Excellence d'Aix-Marseille Université, AMX-19-IET-004) and ANR funding (ANR-17-EURE-0029 to AW), and ANR (LoGiK, ANR-17-CE16-0022 to DD, Plastinex, ANR-21-CE16-013 to DD). We thank A Venture & K Milton for excellent animal care.

## DISCLOSURES:

The authors declare no conflict of interest.

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<figure><img src="image_3.png" /><figcaption>**Figure 1.** Method for obtaining brain slices containing the dLGN. A. Tools necessary for extracting the brain. B. Slicer. C. Cutting of the brain in frontal plans. D. Glue on the</figcaption></figure>
plate. E. Disposition of the brain on the plate. F. Brain glued on the plate. G. Slicing. H. Taking of the slice. J. Slices in the recovery solution.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** View of the slice containing the dLGN and visualization of neurons under the microscope. A. Global view with the objective X4 of the slice containing the dLGN and the vLGN and immobilized in the recording chamber by a nylon thread. B. View with the objective x40 of dLGN neurons in the contralateral region of the dLGN symbolized by the dotted rectangle in panel A.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

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<figure><img src="image_8.png" /></figure>

**Figure 3.** Induction of LTP-IE in a dLGN neuron. A, recording configuration and induction stimulus. Stimulus used for LTP-IE induction consisted of 15 pulses of ~1 nA to elicit an action potential by each depolarizing current pulse. B, time-course of LTP-IE. Top, representative traces before (black) and after (red) LTP-IE induction. Bottom, time-course of the normalized action potential number per depolarizing pulse as a function of time. Note the increased spike number that does not show any decline over >40 min. Adapted from Duménieu et al., BioRxiv 2023. C, stability of input resistance. D, left, group data on 8 dLGN neurons showing the reproducibility of LTP-IE. Right, group data on 8 dLGN neurons showing the lack of R<sub>in</sub> change.