A protocol to investigate cellular and circuit mechanisms generating sharp wave ripple oscillations in rodent basolateral amygdala using ex vivo slices

Abstract

Basolateral amygdala circuits generate oscillatory network activity to process and remember emotion-tagged events. Ex vivo preparations that recapitulate network activities seen in vivo provide an ideal system to investigate the mechanisms driving these network oscillations. Here we describe an ex vivo preparation of basolateral amygdala slices from rodents for measuring the generated sharp wave ripple oscillations (SWs) using local field potential recording and targeted recording from chandelier neurons that initiate SWs. For complete details on the use and execution of this protocol, please refer to Perumal et al. (2021).

Keywords: Cell Biology; Cognitive Neuroscience; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

Preparation of oblique slices containing basolateral amygdala (BLA) and recording for spontaneous sharp-wave ripples (A) Photo of in-house designed storage chamber filled with extracellular solution and bubbled with carbogen delivered through a curved tubing. (B) Schematic of brain glued on its dorsal side on an angled block (10°) and dotted arrows indicate slicing blade movement. Schematic below shows a typical oblique horizonal slice and and boundaries of the BLA highlighted as a triangular region highlighted in red. (C) Top photo of 5× image shows a typical oblique horizontal slice containing BLA. Photo below shows BLA as a triangular region bounded laterally by external capsule and caudally by hippocampus. Scale bar: 100 μm. Inset below shows a representative 20× image of viable BLA neurons. Note the smooth appearance of somata. Scale bar: 50 μm. (D) PNs and INs can be distinguished from intrinsic action potential (AP) discharge characteristics. Top, representative traces from a PN (red) and an IN (blue) showing regular and fast continuous discharge patterns. Bottom left, superimposed APs recorded in a PN (red) and IN (blue) showing that the PN-AP is wider; right, the width at the half-maximal amplitude of AP (AP half-width) in PNs (0.9 ± 2 ms, n=72) was significantly wider than in INs (0.4 ± 0.1 ms, n=54, t-test with Welch correction, p-value < 0.0001). (E) Spontaneous LFP recording in the BLA. Representative image shows extracellular electrode placed in the BLA to record LFP, scale bar: 100 μm. Top trace shows spontaneously occurring deflections in the field potential. Same LFP signal filtered for sharp-wave (1–20 Hz) and ripple (100–300 Hz, bottom) frequencies. Note that each filed potential deflection contain both SW and ripple frequencies. (F) Simultaneous recording for field potential and correlated post-synaptic potentials in a PN (red triangle) and an IN(blue oval). Top trace show LFP signal filtered for ripple frequency band that occur concurrently with inhibitory post-synaptic potentials (IPSPs) in the PN and excitatory post-synaptic potentials (EPSPs) with a burst of action potentials in the IN. (G) LFP and same pair of cells in voltage clamp showing ripple-associated inhibitory post-synaptic currents (IPSC) in the PN (Vh −50 mV; middle) and excitatory post-synaptic currents (EPSC) in the IN going (Vh −70 mV, bottom). Data reported already in Perumal et al. (2021).

Figure 2

Targeted recording from GABAergic interneurons (INs) initiating SW burst (A) Left, photo of oblique brain slice and dots (cyan) indicate approximate location of INs that evoked feedback excitation. Scale bar: 100 μm. Right, top and bottom photos show of triangular and square shaped somata of INs with feedback excitation. Red arrowhead indicates thick bifurcating dendrite. Scale bar:10 μm. (B) Traces show four types of intrinsic action potential discharge patterns elicited by depolarizing suprathreshold current injection in INs with feedback excitation. (C) Top, somata shape among recovered INs with feedback excitation. Bottom, proportion of cells with distinct intrinsic discharge patterns. (D) Whole-cell recording from a fast spiking IN. An action potential evoked with depolarizing current injection (1.5 nA, 1 ms) evoked a time-locked di-synaptic feedback EPSP (red arrow); inset shows mean trace of feedback EPSPs (cyan) overlaid on individual trials. Bottom traces show trials when the action potential evoked a summating EPSP burst, driving time-locked action potentials; a single trial highlighted in cyan is overlaid on individual trials. (E) Voltage-clamp recording (Vh −70 mV) in the IN. A depolarizing voltage step (+70 mV, 0.5 ms) evokes an unclamped 'action potential current' followed by a time-locked burst of feedback excitatory postsynaptic currents (EPSCs, red arrow). The mean trace in cyan overlaid with individual trials. Bottom graph shows distribution of EPSCs in all trials (n=40 trials). Note regular spikes at intervals ∼4 ms following the evoked action potential current at 0 ms. The di-synaptic feedback is highlighted by dotted rectangle. (F) The evoked feedback burst was reversibly blocked by a AMPA receptor antagonist, CNQX (upper) and a GABA_A receptor antagonist, gabazine (lower). Data reported in Perumal et al. (2021).

Figure 3

Axonal arborization and synaptic targeting of chandelier INs in the basolateral amygdala (BLA) (A) Extensive axonal arborization by INs with feedback excitation in the BLA. Reconstruction of three INs (top and bottom left) that evoked feedback excitation with soma and dendrite in blue and axon in red. Same three cells overlaid on a mouse BLA slice with their approximate location. Note individual neurons cover extensive area of BLA. Scale bar: 100 μm. (B) The axonal plexus of INs with feedback contained strings of synaptic bouton ‘‘cartridges’’ in close approximation to the axon initial segment marker Ankyrin G. Top panel, 20× magnified image of recovered axonal plexus from an IN with feedback burst showing multiple strings of synaptic bouton cartridges (red arrows); the inset shows a 100× magnification of a cartridge containing a string of synaptic boutons (red arrows). Bottom panels from the same axon at 100× magnification and panels from left to right show two axon initial segments stained for Ankyrin G (white), biocytin-recovered cartridge synaptic boutons in the same location, and superimposed images of biocytin and Ankyrin G staining exhibits close approximation of cartridge synaptic boutons (red) on Ankyrin G (white). Scale bar: 10 μm. Data reported already in Perumal et al. (2021).