Choking on Inhibition in the Reticular Thalamus

Tenuous Inhibitory GABAergic Signaling in the Reticular Thalamus.

Klein PM, Lu A, Harper ME, McKown HM, Morgan J, Beenhakker MP. J Neurosci 2018;38:1232–1248.

Maintenance of a low intracellular Cl⁻ concentration ([Cl⁻]_i) is critical for enabling inhibitory neuronal responses to GABA_A receptor mediated signaling. Cl⁻ transporters, including KCC2, and extracellular impermeant anions ([A]_o) of the extracellular matrix are both proposed to be important regulators of [Cl⁻]_i. Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl⁻]_i remains relatively low in RT neurons. Although we identify a small contribution of [A]_o to a low [Cl⁻]_i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl⁻. Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl⁻ following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl⁻ extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.

Commentary

As we navigate the world around us, we are barraged by information that we must sense, filter, and attend to. The thalamus, a central and widely connected brain structure, acts as a gateway for this sensory input and shapes the flow of information to and from higher cortical brain structures. The reticular thalamus (RT), a shell of GABAergic neurons that surrounds other thalamic structures, plays an integral role in shaping thalamic activity based on its extensive connectivity both with the cortex and within the thalamus. RT neurons receive co-lateral excitation from both thalamocortical and corticothalamic fibers as well as receive inhibitory inputs from other nearby RT neurons. Dysfunction of RT neurons can lead to altered thalamic oscillatory activity and is associated with absence seizures (1–3), as well as a wide array of neurological dysfunctions, including attentional deficits and hallucinations (4–6). Therefore, it is critical to understand how excitatory glutamatergic and inhibitory GABAergic synaptic input shape neuronal activity in the RT. Here, we focus specifically on the complexities of the GABAergic system in the RT.

Throughout the brain, GABAergic inhibition relies on the flow of chloride (Cl⁻) ions, which are driven across the plasma membrane by the transmembrane Cl⁻ gradient. The intracellular chloride concentration ([Cl⁻]_i) is normally quite low and the transmembrane gradient is quite large. Therefore, when GABA receptors open, Cl⁻ flows into the cell. Under these conditions, the reversal potential for GABA receptors (E_GABA) is hyperpolarizing, driving the membrane potential away from the action potential threshold. When GABA receptors are highly active, however, Cl⁻ can accumulate inside the cell and alter the Cl⁻ gradient. Under these circumstances, E_GABA becomes more depolarized and when GABA receptors open, Cl⁻ ions can flow out of the cell, exciting it (7). The K⁺/Cl⁻ co-transporter KCC2 plays an important role in fighting against Cl⁻ accumulation by pumping Cl⁻ ions out of the cell, lowering [Cl⁻]_i and keeping E_GABA hyperpolarizing (8). Thus, the activity of KCC2 is essential to maintaining inhibitory drive in the face of ongoing GABAergic activity.

In this study by Klein and colleagues, the Beenhakker Lab probed how GABAergic inhibition is maintained in RT neurons by the K⁺/Cl⁻ co-transporter KCC2. This study developed from an interesting and important observation that immunolabeling for KCC2 is notably low in the reticular thalamus. Does this relative lack of KCC2 mean that RT neurons have depolarizing E_GABA? Interestingly, Klein and colleagues found that even the low levels of KCC2 present in the RT function to keep GABA neurotransmission hyperpolarizing. First, they demonstrated with perforated patch recordings that even though KCC2 levels are low, E_GABA is ≈−65 mV in RT neurons. This means that GABAergic activation will drive RT neurons away from action potential threshold. Second, they showed with calcium imaging that the activation of GABA_A receptors, via muscimol, decreased RT neuron activity. Third, they showed that application of a KCC2 antagonist, VU0463271, resulted in a time-dependent increase in E_GABA in RT neurons of ≈12 mV. Finally, they demonstrated that blockade of KCC2 resulted in muscimol-driven activation of neuronal activity, a reversal of muscimol's effects when KCC2 is active. Together, these studies demonstrate that even the low levels of KCC2 present in RT neurons are critical for maintaining hyperpolarizing responses to GABA. The authors deserve to be commended for carrying out such a rigorous and technically challenging set of experiments.

What, then, is the functional consequence of the low levels of KCC2? It appears that without abundant levels of KCC2, RT neurons struggle to maintain low [Cl⁻]_i when synaptic inhibition is increased. During times of heightened GABAergic activity, large Cl⁻ influxes occur, and the minimal amount of KCC2 present in RT neurons simply cannot pump Cl⁻ out fast enough. In a series of experiments, the authors show that GABA-induced increases in [Cl⁻]_i recover slowly in RT cells. This is presumably due to the relative lack of KCC2, which was confirmed by showing that the rate of recovery of E_GABA in RT neurons was unaffected by pharmacologically inhibiting KCC2. In VB neurons, however, which express KCC2 at greater levels, [Cl⁻]_i recovers more rapidly after heightened GABAergic activity, and the rate of recovery is strongly slowed by inhibition of KCC2. These findings squarely pinpoint a role for the decreased levels of KCC2 in the RT: When GABAergic activity occurs, RT neurons are “tenuously” close to sliding into GABAergic excitation. This may be especially important as RT neurons receive significant GABAergic input from other nearby RT neurons. As excitation from thalamocortical and corticothalamic axons drives RT neurons to inhibit each other, the summation of GABAergic inputs can rapidly increase [Cl⁻]_i. In the RT, where KCC2 is low, this means that GABAeric activity can rapidly transition from inhibition to excitation. To examine this, the authors employed a computational model to show that GABAergic synaptic transmission between RT neurons can rapidly become excitatory and lead to widespread activation of the entire RT network.

In addition to examining KCC2's role in controlling [Cl⁻]_i, the authors also probe the role of impermeant anions in RT, which have recently been shown to contribute to E_GABA (9). Chondroitin sulfate proteoglycans (CSPGs) are highly charged extracellular molecules that are enriched in the reticular thalamus. Do these highly charged extracellular molecules contribute to E_GABA in RT neurons? By degrading these proteins in acute brain slices with the enzyme ChABC, the authors showed that CSPGs do contribute to E_GABA—but only minimally. Interestingly, their results demonstrated that enzymatic degradation of extracellular impermeant anions only changes E_GABA after prolonged periods of stimulation. These studies underscore the importance of KCC2 function in RT, as the high levels of CSPGs do not appear to provide significant control over chloride homeostasis in the RT.

Does the tendency to shift from hyperpolarizing to depolarizing GABA engender RT with a special set of properties? First, it likely helps make the thalamus a powerful rhythm generator. The dynamic nature of GABAergic signaling perhaps enables the coherent activation of a small group of cells embedded in an inhibitory network, leading to thalamic rhythms critical to sleep, attention, and sensory processing. Second, it puts the RT dangerously close to generating pathological oscillatory activity associated with epilepsy. Indeed, attenuation of inhibition in the RT, by pharmacological inhibition of GABA receptors (10) or knock-down of the β3 subunit of the GABA receptor (1), enhances and prolongs thalamic oscillatory activity. In reading this paper, one might struggle to understand how inhibition can persist in the RT at all! So many inhibitory connections exist in the RT, and activity levels can be quite high. Readers can put their minds at ease though, as in vivo RT cells maintain a membrane potential near −60 mV (11). So even though GABAergic inhibition is tenuous, RT neurons remain hyperpolarized, and thalamic activity does not spiral out of control under normal conditions. In summary, this study from the Beenhakker Lab represents a significant step forward in understanding the dynamic nature of GABAergic neurotransmission in the RT and how it shapes thalamic activity.