GABA neurons in the ventral tegmental area of the midbrain are important components in the brain's reward circuit. Long term changes in this circuit occur through the process of synaptic plasticity. It has been shown that high frequency stimulation, as well as treatment with endocannabinoids, can cause GABA neurons in the ventral tegmental area to undergo long term depression, a form of synaptic plasticity that decreases excitability of cells. The present study elaborates on the mechanism whereby high frequency stimulation can result in long term depression of ventral tegmental area GABA neurons. Using the whole cell patch clamp technique in acute brain slices, we recorded excitatory currents from ventral tegmental area GABA neurons in GAD-GFP expressing CD1 mice and observed how the excitatory currents changed in response to different treatments. We confirm that high frequency stimulation causes long term depression, and the cannabinoid type 1 receptor antagonist AM-251 blocks this effect. Long term depression is also elicited by treatment with the cannabinoid type 1 receptor agonist 2-arachidonylglycerol. It is inconclusive whether treatment with 2-arachidonylglycerol occludes further long term depression by high frequency stimulation. We also demonstrate that activation of group I metabotropic glutamate receptors by DHPG produces long term depression. These results support the model that at these excitatory synapses, high frequency stimulation causes the release of glutamate from presynaptic terminals, activating group I metabotropic glutamate receptors, causing production of 2-arachidonylglycerol. 2-arachidonylglycerol in turn acts on presynaptic cannabinoid type 1 receptors to decrease release of glutamate onto GABA neurons. This model can be tested by further research, which should include cannabinoid type 1 receptor knockout mice. This study provides more insight into how drugs of abuse such as tetrahydrocannabinol, the active component of marijuana that activate cannabinoid type I receptors, can corrupt the natural reward mechanisms of the brain.

Endocannabinoids (eCBs) are lipophilic signals that are produced by postsynaptic neurons in an activity-dependent manner, and signal in a retrograde fashion to modulate neurotransmitter release. As such, eCBs are highly involved in synaptic plasticity, a process that strengthens or weakens synapses. eCB-mediated synaptic plasticity is involved in many brain processes including learning, short-term memory, and adaptive reward, which are processed in the hippocampus and ventral tegmental area (VTA), respectively. However, the expression of eCB biosynthetic enzyme mRNA within hippocampal and VTA neurons, as well as the relationship between these mRNA species and the occurrence of synaptic plasticity, remains unclear. The goal of these studies was to demonstrate the expression pattern of eCB biosynthetic enzyme mRNA within hippocampal and VTA neurons, and to describe the relationship between synaptic plasticity and mRNA expression. Using whole-cell electrophysiology and real-time quantitative PCR, I tested hippocampal and VTA neurons for the presence of eCB biosynthetic enzyme mRNA and described the relationship between these enzymes and synaptic plasticity. The data presented herein demonstrate the importance of eCB signaling within the hippocampus and VTA and the expression patterns of eCB biosynthetic machinery within several neuron types. These data provide evidence that eCB signaling plays a critical role in learning, short-term memory, and adaptive reward.

Adults exhibit significant differences in drug vulnerability, learning ability, and emotive processing than adolescents. Long-lasting synaptic changes, including long-term depression (LTD), can endure many hours and are believed to be key to encoding memories and persistent cognitive changes such as addiction. The ventral tegmental area (VTA) is the primary source of midbrain dopamine (DA) and is regulated by local inhibitory GABA neurons. GABA regulation can decrease, resulting in dis-inhibition of DA neurons and increased feelings of reward, learning, or salience attachment to memories. Endocannabinoids (eCBs) are signaling molecules that often result in synaptic plasticity changes, and the eCB system has shown pronounced cross-talk with opioid signaling and receptor pathways. We examined eCB mediated plasticity of excitatory inputs to GABA neurons within the VTA and how THC, opioids, and age influence this plasticity. We determined that direct CB1 receptor activation in the mouse results in reduced excitatory input activity to GABA, resulting in GABA depression. CB1-mediated GABA depression was lost following chronic exposure to THC, suggesting THC injection has already activated this pathway. Chronic THC occlusion of GABA LTD was then reversible following a week of drug withdrawal. Next, as adult animals tend to exhibit reduced emotive influence, learning, and drug affect when compared to adolescents we examined if LTD of VTA GABA neurons was present in adults. We determined that adult mice no longer undergo HFS-induced LTD. Interestingly, the eCB pathway is still active, as we determined that both CB1 receptor activation and mGluR5 activation still results in GABA depression in adults. We then determined that a greater electrical HFS could induce LTD of excitatory inputs to adult VTA GABA neurons, suggesting a change in adults leading to an increase in induction thresholds for GABA plasticity. Morphine was found to induce similar LTD of GABA neurons through the mu-opioid receptor in both adolescents and adults. This LTD is likely pre-synaptic, similar to THC induced LTD, and is potentially mediated through the same presynaptic pathways as CB1-dependent LTD as injection of either THC or morphine eliminates depression by the other. Chronic morphine injection eliminates HFS-induced LTD in adolescents but only results in a loss of LTD in 58% of adult experiments suggesting resistance to morphine exposure at this synapse in adults. In summary, THC and morphine appear to act pre-synaptically to induce GABA LTD within the VTA and occlude further LTD by the other drug. Additionally, development into adulthood significantly alters the propensity of GABA neurons to undergo plasticity and greater levels of stimulus are required to elicit lasting changes.

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 m multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4–5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

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