Ketamine Reverses Lateral Habenula Neuronal Dysfunction and Behavioral Immobility in the Forced Swim Test Following Maternal Deprivation in Late Adolescent Rats

Abstract

Mounting evidence suggests that the long-term effects of adverse early life stressors on vulnerability to drug addiction and mood disorders are related to dysfunction of brain monoaminergic signaling in reward circuits. Recently, there has been a growing interest in the lateral habenula (LHb) as LHb dysfunction is linked to the development of mental health disorders through monoaminergic dysregulation within brain reward/motivational circuits and may represent a critical target for novel anti-depressants, such as ketamine. Here, we show that maternal deprivation (MD), a severe early life stressor, increases LHb intrinsic excitability and LHb bursting activity, and is associated with the development of increased immobility in the forced swim test (FST) in late-adolescent male rats. A single in vivo injection of ketamine is sufficient to exert prolonged antidepressant effects through reversal of this early life stress-induced LHb neuronal dysfunction and the response in the FST. Our assessment of ketamine's long-lasting beneficial effects on reversal of MD-associated changes in LHb neuronal function and behavior highlights the critical role of the LHb in pathophysiology of depression associated with severe early life stress and in response to novel fast-acting antidepressants.

Keywords: LHb; depression; early life stress; forced swim test; intrinsic excitability; ketamine; lateral habenula; maternal deprivation.

Figure 1

Ketamine normalized maternal deprivation (MD)-induced behavioral changes in forced swim test (FST) in adolescent rats. (A) Upper panel demonstrates the time course of behavioral transition from active (swimming and climbing) to passive (immobility) coping behaviors. Both non-MD and MD late-adolescent rats quickly learned to adopt immobility during the first swim session (Pre-test, non-MD: n = 18, MD: n = 26; climbing time course: F_(2,72) = 107.3, ****P < 0.0001; swimming time course: F_(2,72) = 39.72, ****P < 0.0001; immobility time course: F_(2,72) = 11.4, ****P < 0.0001; two-way ANOVA). Lower panel shows that MD induced a significant decrease in climbing behavior and a significant increase in immobility in FST (Test, non-MD + saline: n = 10, non-MD + Ketamine: n = 8, MD + saline: n = 13, MD + Ketamine: n = 13; swimming: *P < 0.05; climbing: **P < 0.01; immobility: **P < 0.01). Administration of ketamine (20 mg/kg) significantly decreased MD-induced immobility 24 h post-injection (swimming: F_(1,40) = 8.71, **P < 0.01; immobility: F_(1,40) = 6.27, *P < 0.01; two-way ANOVA). (B) Upper panel demonstrates the adoption of immobility in juvenile rats during Pre-test. Similar to late-adolescent rats, non-MD and MD juvenile rats quickly learned to adopt immobility during the first swim session (Pre-test, n = 14 in each group; climbing time course: F_(2,52) = 175.4, ****P < 0.0001; swimming time course: F_(2,52) = 82.48, ****P < 0.0001; immobility time course: F_(2,72) = 23.95, ****P < 0.0001; two-way ANOVA). Lower panel shows that MD induced a significant increase in climbing behavior and a significant decrease in immobility in the FST (Test, n = 7 in each group, climbing: *P < 0.05; immobility: *P < 0.05) in juvenile rats (P21–P28). Administration of ketamine (20 mg/kg) attenuated MD-induced changes in climbing and immobility 24 h post-injection (Test, climbing: F_(1,24) = 26.69, ****P < 0.0001; immobility: F_(1,24) = 18.76, **P < 0.01; two-way ANOVA). All data is represented as means ± SEM.

Figure 2

Ketamine normalized MD-induced changes in lateral habenula (LHb) intrinsic excitability and firing patterns in late-adolescent rats. (A) Distribution of LHb neurons based on firing patterns in whole cell current-clamp recordings of spontaneous activity. The insets show example traces from LHb neurons with silent, tonic and bursting patterns recorded from non-MD or MD rats that received either saline or ketamine injections (recorded 72 h post-injection). The number of cells/rats are similar to the ones reported in (D). MD increased spontaneous LHb neuronal activity (specifically bursting) and ketamine reversed these effects of MD. (B) Representative traces from LHb neurons in non-MD and MD rats that received either saline or ketamine (20 mg/kg) injections and sacrificed after 72 h. (C) Figure shows the average of whole cell action potential (AP) recordings in response to depolarizing current injections (I) with intact synaptic transmission in LHb slices from P42 to P50 non-MD and MD rats. MD did not alter LHb neuronal excitability with intact synaptic transmission. (D) Whole cell patch clamp recordings of action potentials (APs) in response to depolarizing current injections (I) with blocked synaptic transmission from LHb neurons in non-MD and MD rats that received either saline or ketamine (20 mg/kg) injections and sacrificed after 72 h. MD increased LHb intrinsic excitability and ketamine reversed this effect (F_(3,360) = 15.84, ****P < 0.0001; two-way ANOVA). (E) Measurements of intrinsic properties of LHb neurons including input resistance (Rin), fast afterhyperpolarization (fAHP), medium afterhyperpolarization (mAHP) and AP threshold. Average amplitude of fAHP, mAHP and Rin were generated from recordings in (D). MD significantly increased Rin and ketamine reversed this effect of MD (F_(1,33) = 3.936, *P < 0.05; two-way ANOVA). Numbers indicated in (C,D) represent the number of cells recorded per rats in each group.