To study the cellular basis of behavioral depression we examined the synaptic circuitry in the LHb of rats displaying learned helplessness (LH), a model of depression whereby animals show reduced escape from escapable foot shock10
. We employed two well-established animal models: acute learned helplessness (aLH), which is induced by subjecting rats to periods of inescapable and unpredictable shock (aLH)10
; and congenital learned helplessness (cLH), a strain of rats produced by selective breeding of animals displaying the greatest amount of aLH11, 12
. In addition to showing reduced escape from escapable footshock, cLH and aLH animals also showed increased immobility in the forced swim test, another widely used animal model for depression13
, compared with control animals ().
Enhanced synaptic transmission onto VTA-projecting LHb neurons correlates with helpless behavior of individual animals
We examined transmission onto LHb neurons, which receive major inputs from numerous brain regions involved in stress response (such as the entopeduncular nucleus, lateral hypothalamus, lateral preoptic area, medial prefrontal cortex, and the bed nucleus of the stria terminalis; Supp Fig. 1
) and can control dopaminergic function in the midbrain4
. We wished to determine if synaptic transmission onto LHb neurons is different in LH compared to normal animals. To record selectively from LHb neurons that can regulate dopamine neuron activity, we injected in vivo
Alexa 488 conjugated Cholera toxin, a retrograde tracer, in the VTA. Two to three days later, we prepared brain slices that contained the LHb. A minority of neurons in the LHb displayed fluorescent cell bodies, which indicated their projection to the VTA (Supplemental Fig. 2 a & b
). Notably, the LHb neurons that project to VTA and RMTg, a newly identified GABAergic relay station between LHb and VTA15, 16
, form largely non-overlapping populations (Supplemental Fig. 2 c
), indicating that we were able to selectively target LHb neurons directly projecting to the VTA. LHb neurons projecting to the VTA were glutamatergic, as indicated by their co-localization with the glutamate transporter EAAC1 and lack of GABAergic marker expression (Supplemental Fig. 3
). We performed whole-cell patch clamp recordings on VTA-projecting neurons in acute parasagittal brain slices prepared from rats that were wild type control, aLH, cLH (naïve), or cLH rats exposed to mild stress (cLHms, see Behavioral Paradigms in Methods). We examined miniature excitatory postsynaptic currents (mEPSCs, in the presence of tetrodotoxin to block action potentials and picrotoxin to block GABA-A-mediated synaptic currents), which were mediated by AMPA-type glutamate receptors (Supplemental Fig. 4 a
) and represent responses from individual synapses onto these cells. The mean frequency of mEPSCs recorded from VTA-projecting LHb neurons of aLH (3.7 ± 0.8, mean ± s.e.m., n=23), cLH (4.0±0.7, n=85) and cLHms (4.7 ± 0.7, n=70) rats was increased relative to WT controls (2.3 ± 0.2, n=82; F(3,251) = 3.1, p < 0.03, ANOVA; ). In general, the distribution of mEPSC frequencies recorded across different cells in all groups displayed a bimodal distribution (). Notably, the prevalence of neurons with high-frequency mEPSCs (>8 Hz; shaded region, ) was significantly higher in aLH (17%), cLH (14%) and cLHms (20%) compared to WT (2%; p< 0.01, c2
). To determine if the observed excitatory synaptic potentiation was quantitatively correlated to an animal’s helpless behavior, we first tested animals (either WT or cLH) with an escape avoidance task, and subsequently prepared brain slices and recorded from VTA-projecting neurons. From each animal we recorded from at least five cells and plotted the average mEPSC frequency against the animal’s helpless behavior (as measured by the fraction of trials the animal failed to escape from an escapable 10 sec foot shock; also see ). The significant correlation (; R2
= 0.69, F(1, 11)=24.85, p< 0.001, n = 13 for all animals; R2
=0.64, F(1,6)=10.7, p<0.05, n=8 for cLH animals only) indicates that the potentiation of excitatory transmission onto VTA-projecting LHb neurons is linked with an individual animal’s helpless behavior. To examine the output of VTA-projecting neurons we measured their spontaneous action potentials, which were more frequent in cLH animals compared to WT controls (). We observed no differences among the various groups with respect to the amplitude of mEPSCs (), frequency or amplitude of miniature inhibitory postsynaptic currents (supplemental Fig 4 b, c
). These results indicate that the excitatory synaptic input onto LHb neurons that project to the VTA is potentiated in the learned helplessness model.
Increased excitatory synaptic transmission onto VTA-projecting LHb neurons in the learned helplessness models of depression
The enhanced mEPSC frequency could be due to an increase in either the number of synapses, or the probability of presynaptic neurotransmitter release. To distinguish between these possibilities, we first measured the density of dendritic spines, sites of excitatory synapses, on the dendrites of VTA-projecting LHb neurons. There was no significant difference in spine density between the WT controls and cLH animals (), and there was also no obvious difference in the dendritic branching between the two groups (not shown), suggesting there was no major difference in the number of synapses between control and cLH animals.
Presynaptic mechanism underlying the increase in excitatory synaptic transmission onto VTA-projecting LHb neurons in helpless animals
To determine whether there is a change in the efficacy of presynaptic neurotransmitter release, we examined evoked transmission. Synaptic transmission onto LHb neurons (elicited by placing a stimulating electrode in the LHb) showed distinct properties: the evoked excitatory synaptic response had a very small NMDA receptor component (Supplemental Fig. 4 d
) and the AMPA receptor component displayed strong inward rectification (Supplemental Fig. 4 e
). To probe presynaptic function we evoked transmission with high frequency stimulation trains (10 stimuli delivered at 20 or 50 Hz). The decrease in the amplitude of EPSCs in response to successive pulses during a train of stimuli reflects presynaptic vesicle depletion; more depletion correlates with higher release probability17
. VTA-projecting LHb neurons of cLH animals showed a faster (20 Hz: F(9, 198)=2.32, p<0.05; 50 Hz: F(9, 198)=3.83, p<0.001) and more extensive (20 Hz: F(1, 22)=6.62, p<0.05; 50 Hz: F(1, 22)=7.15, p<0.05; One-Way ANOVA with repeated measures) synaptic depression, compared with that in WT control animals (). Furthermore, with minimal stimulation, which is designed to activate few synapses (as indicated by the amplitude of non-failure responses that is similar to the mEPSC amplitude; ), we measured synaptic transmission failure rate. Excitatory synaptic transmission onto VTA-projecting LHb neurons of cLH animals had a significantly lower failure rate compared with that of control animals (; n=7–9, p <0.001, bootstrap). These results indicate that the excitatory synaptic inputs onto VTA-projecting LHb neurons of helpless animals have a higher synaptic release probability; therefore repeated stimulation can deplete synaptic vesicles faster and more efficiently in helpless animals.
One treatment for clinical depression, currently under evaluation, is deep brain stimulation (DBS), consisting of continuously delivered high frequency electrical stimulation to various brain regions8, 18
. In a recent clinical case, DBS of LHb produced a marked remission of treatment-resistant depression9
. Notably, depression recurred when DBS was stopped (in two accidental episodes9
). To examine the cellular effects of DBS, we recorded in brain slices synaptic transmission onto VTA-projecting LHb neurons evoked by placing a stimulating electrode in the LHb. After a baseline period of evoked transmission, a DBS protocol used in patients (7 stimulus 130 Hz trains separated by 40 ms intervals) was continuously delivered through the same stimulation electrode; stimuli interleaved with the DBS trains permitted us to monitor evoked synaptic transmission (see details in methods). The DBS protocol produced a marked depression of excitatory synaptic transmission, which persisted for the DBS protocol period, and was reversed upon cessation of the DBS protocol (). Thus, a DBS protocol can effectively reduce excitatory synaptic transmission onto VTA-projecting LHb neurons.
DBS in LHb suppresses excitatory synaptic transmission and reverses learned helplessness
We wished to test if reducing synaptic drive onto LHb neurons can modulate helpless behavior. Remarkably, the same DBS protocol as used in brain slices delivered to the LHb in aLH animals markedly ameliorated the helpless behavior as indicated by an increase in escape behavior (; Supplemental Fig. 5 a
). This effect was dependent on both the intensity of stimulation, and the placement of the stimulation electrode: stimulating at 300 µA had a stronger behavioral effect and affected a larger volume within the LHb than stimulating at 150 µA (, and Supplemental Fig. 5 b & c
); and only if the electrode was placed in the lateral habenula, but not in the nearby thalamus, did DBS reverse the helplessness (). Furthermore, DBS in LHb, but not sham stimulation in LHb, prevented the increase in immobility in the forced swim test (). Thus, suppression of synaptic transmission at the LHb through DBS can acutely reverse helpless behavior in rats.
A number of changes in neural function have been identified in depressed humans and rodent models of depression, likely due to the multifaceted nature of depressive disorders.19, 20, 21, 22, 23
The recent identification of the LHb as a brain region in monkeys that can encode disappointment and expectation of negative conditions2, 3
led us to investigate its role in the learned helplessness rodent model of depression. Our findings indicate that excitatory synaptic activity onto VTA-projecting neurons in the LHb may be a key modulator of learned helplessness. The two learned helplessness models examined showed potentiated excitatory synaptic activity onto these neurons. Interestingly, the major modification was an increase in the proportion of cells displaying high-frequency mEPSCs in LH animals, from 2% to 14–20%. This suggests that large changes in a small proportion of cells in the LHb may be capable of modifying behavior. A critical role for transmission onto LHb neurons is further supported by the strong correlation between the potentiation of synaptic transmission onto VTA-projecting LHb neurons and an individual animal’s helpless behavior. Given the presynaptic nature of synaptic potentiation, we examined the effects of synaptic depression by repeated afferent stimulation, a protocol that mimics clinically used DBS. Reducing synaptic transmission onto LHb neurons through a DBS protocol led to acute reversal of learned helplessness. Suppression of transmission onto VTA-projecting LHb neurons likely played a role in mediating this beneficial effect, although modulation of LHb neurons, or axons of passage, projecting to other targets may also be involved.
Our study provides cellular mechanisms that may explain previously reported phenomena: a) an increased LHb metabolic activity observed in depressed humans24, 25
and animal models of depression26, 27
, and b) lesion28, 29
or pharmacological silencing30
of the LHb can modulate depression-like symptoms in animal models. Our findings suggest an aberrant cellular process previously unexamined in the context of mood disorders that may be critical in the etiology of depression. Future studies aimed at determining the molecular signaling changes underlying the synaptic hyperactivity onto LHb neurons may lead to novel and effective treatments potentially able to reverse some forms of depressive disorders.