Abstracts

This abstract is a recipient of the Young Investigator Award
This abstract has been invited to present during the Basic Mechanisms platform session

Rationale: Current literature views lactate as a waste, end-product of the anaerobic glycolysis. Recent studies, however, suggest that lactate can act as a brain signaling molecule via lactate receptor, hydroxycarboxylic acid receptor type 1 (HCA₁R). Epileptic seizures accelerate glycolysis to restore energy and lead to lactate accumulation. Here we investigated whether extracellular lactate can modulate seizure activity and neuronal excitability in vivo and in vitro.

Methods: We measured in vivo extracellular lactate concentration during a seizure and status epilepticus (SE) using a lactate biosensor probe with a simultaneous EEG recording. To study the role of HCA₁R during seizures, we induced SE and a rapid kindling protocol in HCA₁R KO and littermate wild-type (WT) control mice. We investigated the effect of activation of HCA₁R in vitro by using genetically-encoded calcium indicator GCaMP7. Briefly, we transfected HCA₁R KO and WT mice with a mix of CamKIICre and GCaMP7 into CA1 hippocampus. We studied GCaMP7-transfected CA1 principal neurons in vitro under wide-field microscopy following perfusion with lactate or HCA₁R agonist, 3-chloro-5-hydroxybenzoic acid (3CL-HBA). Active and passive neuronal membrane properties from CA1 principal neurons were assessed using patch-clamp electrophysiology. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in a voltage clamp. Evoked excitatory postsynaptic currents (eEPSCs) were recorded in response to stimulation of Schaffer collateral axons.

Results: Extracellular lactate concentration rose quickly during a seizure and SE reaching millimolar concentration rage (0.777mM (±0.407), n=6, p=0.02, and 0.957mM (±0.176), n=5, p=0.03, paired t-test, respectively), which is sufficient to activate HCA₁R. HCA₁R KO mice were more susceptible to being kindled (HCA₁R KO: n=6; HCA₁R WT: n=7; p=0.02, Fisher's test) and SE (HCA₁R KO n=9; HCA₁R WT n=16; p=0.02, Fisher's test). Moreover, HCA₁R KO mice developed longer (n=9; p=0.03, Kaplan-Meier survival comparison) and more severe SE (n=6; p< 0.0001, 2-tailed Mann–Whitney test) than WT mice. In in vitro GCaMP7 imaging, lactate perfusion decreased the excitability of CA1 principal neurons (n=4 p< 0.0001, One-Way ANOVA). 3CL-HBA, reduced the firing of CA1 neurons in HCA₁R WT but not in KO mice (n=4, p< 0.0001, t-test). In patch-clamp recordings, both lactate (n=9 control; n=5 lactate; p=0.03, unpaired t-test) and 3CL-HBA (n=12 cells; p=0.04, paired t-test) hyperpolarized membrane potential of CA1 pyramidal neurons. HCA₁R activation with lactate (HCA₁R WT: n=4 cells, p=0.03; HCA₁R KO: n=7 cells, p=0.84, paired t-test) reduced the sEPSC frequency and altered the paired-pulse ratio of eEPSCs (n=5 cells, HCA₁R WT: tau=47 vs tau=31; HCA₁R KO: tau=29 vs tau=34) in HCA₁R WT but not in KO mice, suggesting that it reduces glutamate release from the presynaptic terminals.

Conclusions: Excessive neuronal activity accelerates glycolysis to generate lactate, which translocates to the extracellular space to slow neuronal firing and excitatory neurotransmission via HCA₁R. These studies may identify novel anticonvulsant target and seizure termination mechanisms. 

Funding: RO1 NS040337 to JK, UVA Brain Institute