Inducing Long-Term Plasticity of Intrinsic Neuronal Excitability in Neurons of the Dorsal Lateral Geniculate Nucleus

Summary

Here, we describe 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 traveling 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. Here, we report 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 for 10 min. LTP-IE in dLGN relay neurons is long-lasting as it can be followed up to 40 min after the induction protocol. In conclusion, the results of this study provide the first evidence for the induction of intrinsic plasticity in dLGN relay cells, thus further pointing to the role of thalamic neurons in activity-dependent visual plasticity.

Introduction

The overall goal of this method paper is to provide a simple way to induce long-lasting plasticity of neuronal excitability in visual thalamic neurons of the rat in vitro, using the standard current-clamp mode of the patch clamp technique^1,2. The rationale behind the development of this technique is its simplicity and reproducibility. The advantage 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 information³. However, this initial conclusion has been challenged by recent works indicating that this simplistic view does not hold longer^4,5,6. For instance, functional deficits in visual response are already observed in amblyopic patients at the stage of the LGN⁷. In addition, a large proportion of dLGN relay neurons in a given monocular territory receive inputs from each eye, indicating potential binocularity for a large proportion of dLGN neurons^8,9,10. Moreover, monocular deprivation produces a 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^15,16,17,18, spiking activity^19,20, sensory stimulation²¹ or following activity or sensory deprivation^20,22,23. Here, we describe 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^24,25.

Protocol

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)).

1. Animals

1. Conduct experiments using 19-25-day-old Long Evans rats of both sexes (weighing between 50-90 g).
2. House the animals in conventional plastic cages, together with their mother and their littermate. Keep the temperature constant with a 12 h light/dark cycle and ad libitum access to water and food.

2. Acute slices of rat dLGN

1. Prepare cutting/recovery and extracellular recording solutions.
   1. To prepare the cutting/recovery solution, mix 92 mM n-methyl-D-glutamine, 30 mM NaHCO₃, 25 mM D-glucose, 10 mM MgCl₂, 2.5 mM KCl, 0.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 20 mM Hepes, 5 mM sodium ascorbate, and 3 mM sodium pyruvate and bubbled with 95% O₂-5% CO₂ (pH 7.4). Keep the cutting solution cold during the slicing procedure.
   2. Prepare the recording solution (artificial cerebrospinal fluid, ACSF) by mixing 125 mM NaCl, 26 mM NaHCO₃, 2 mM CaCl₂, 2.5 mM KCl, 2 mM MgCl₂, 0.8 mM NaH₂PO₄, and 10 mM D-glucose and permanently equilibrate it with 95% O₂-5% CO₂.
2. Prepare the dissecting tools and two ice platforms (Figure 1A).
3. Put ice in the outer tank and fill the slicing chamber of the vibratome with an ice-cold cutting solution (Figure 1B).
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. Place the head on the first iced platform and water the isolated tissue every 10 s with the oxygenated ice-cold slicing solution.
5. Cut the scalp in a 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.
   NOTE: The time should not exceed 2 min from the decapitation to the extraction of the brain.
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.
7. Glue the brain block on the plate of the vibratome with the rostral side up (see Figure 1D,E). Water the brain regularly (every 10 s) during the whole procedure until it is submerged in the slicing chamber of the vibratome.
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.
9. Aspire the slices with a broken Pasteur pipette connected to a flexible rubber sucker (Figure 1H).
10. Let the slices recover for 20-30 min in the recovery solution (Figure 1I).

3. Whole-cell patch-clamp electrophysiology

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

1. Turn on the pumps of the rig to make the extracellular recording saline (see composition in step 2.1) circulating in the recording chamber.
2. Prepare the intracellular solution by mixing 120 mM K-gluconate, 20 mM KCl, 10 mM Hepes, 0.5 mM EGTA, 2 mM MgCl₂, 2 mM Na₂ATP and 0.3 mM NaGTP (pH 7.4).
   NOTE: Intracellular solution can be prepared in advance and kept frozen at -80°C. When unfrozen, the solution is kept at 4 °C.
3. Add the GABA_A channel blocker, picrotoxin (100 µM final concentration), and the ionotropic glutamate receptor antagonist, kynurenate (2 mM, final concentration), to the extracellular saline.
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.
5. Place the U-shaped platinum wire on the slice and position the slice to visualize the dLGN (Figure 2A).
6. Prepare patch pipettes from borosilicate glass tubes pulled with a puller. Ensure that the patch pipettes have a resistance of 5-10 MΩ when filled with the intracellular solution.
7. Visualize the dLGN at high magnification (40x or 60x) and select a neuron for patch-clamp recording using differential interference contrast infrared video-microscopy (Figure 2B). Select the neurons based on their healthy appearance (i.e., slightly swollen with sharp membrane contours).
8. Position the patch-pipette on the selected neuron with constant positive pressure using the micromanipulator.
   1. 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.
   2. Set the amplifier on CC mode, balance the bridge to compensate for access resistance, and hold the neuron at -65 mV.

4. Data acquisition

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.
2. Monitor neuronal excitability during a control period of about 10 min by injecting a 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.
3. Monitor the input resistance of the neuron throughout the experiment with a brief negative pulse of current (typically -20 pA during 100 ms). Keep the amplitude of the current pulse constant before and after the induction of LTP-IE.

5. Induction of LTP-IE

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 for 10 min.
2. Choose the amplitude of the current pulse to elicit a single action potential each time (i.e., 15 action potentials per train).
   NOTE: Each train of 15 spikes is elicited at a frequency of 0.1 Hz (i.e., 60 trains and 900 spikes).

6. Data analysis

NOTE: The analysis procedure is the same before (control conditions) and after LTP-IE induction.

1. For each recorded trace, calculate the input resistance (R_in) from the 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, discard the experiment.
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.
3. Quantify LTP-IE over a period of 10 min, 20 min after the beginning of the post-induction period by measuring the number of AP evoked by each current pulse and averaging each minute by using a homemade routine in a data analysis software ( refer to Supplementary File 1 for Igor Pro routine). Detect spikes when the voltage signal crosses 0 mV.
   NOTE: LTP-IE is expressed as the number of action potentials 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.

Results

dLGN neurons were recorded in whole-cell configuration, and LTP-IE was induced by action potential firing at 40 Hz for 10 min in the presence of ionotropic glutamate and GABA receptor antagonists (Figure 3A). A three-fold increase in the number of action potentials was observed 20-30 min after the induction (Figure 3B) without any change in input resistance, R_in (Figure 3C). This protocol reliably induced LTP-IE in dLGN n...

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...

Materials

Name	Company	Catalog Number	Comments
Automated vibrating blade microtome	Leica	VT-1200S	vibratome/slicer
Borosilicate glass tube	Phymep	B-15086-10
Controller Typ V	Luigs&Neumann	Typ V	temperature controller
Igor software	wavemetrics		analysis software
Infrared videomicroscopy	Olympus	XM-10 Camera
Kynurenate	Merk/Sigma	K3375	AMPA/NMDA receptors blocker
Low-noise Data Acquisition System	Axon - Molecular devices	Digidata 1440A	analog/digital interface
Micromanipulators	Luigs&Neumann	LN Mini25
Multiclamp 200B	Axon - Molecular devices	N/A	Patch-clamp amplifier
Multiclamp 700B	Axon - Molecular devices	N/A	patch-clamp amplifier
PC-100 puller	Narishige	PC-100	micropipette puller
PClamp10	Axon - Molecular devices	N/A	patch-clamp recording software
Picrotoxin	AbCam	ab120315	GABAA receptors blocker
Slice mini chamber	Luigs&Neumann	LN Chambre Slice mini I-II	Submerged Chamber
Upright microscope	Olympus	BX51 WI