Neurotensinergic modulation of glutamatergic neurotransmission in VTA neurons

The ventral tegmental area (VTA) contains a high density of neurotensin (NT) terminals and receptors that are expressed on dopaminergic (DA) and non-DA neurons. This area of the brain is strongly implicated in disorders like schizophrenia, psychosis and drug abuse. Drugs of abuse induce behavioural sensitization- a facilitatory process whereby exposure to a stimulus results in an enhanced response to a subsequent exposure of the same stimulus. Sensitization develops in the VTA and involves glutamatergic neuroadaptations in VTA DA neurons. NT antagonists prevent the development of sensitization and reward mechanisms and this could be mediated through a modulation of glutamatergic neurotransmission in the VTA. However, how NT modulates glutamatergic neurotransmission in VTA neurons remains unclear.  The present thesis was aimed at investigating the NTergic modulation of glutamatergic neurotransmission in VTA DA and non-DA neurons. Whole cell patch clamp electrophysiology in acute VTA horizontal slices was used to study the effects of different NTergic agonists on VTA neurons.  Neurons were classified as either Ih+ (putative dopaminergic neurons) or Ih- (putative non-dopaminergic neurons) based on the presence or absence of a hyperpolarisation activated cationic current (Ih) respectively. Immunohistochemical techniques were routinely used to label neurons and confirm their location in the medial VTA.  In the first study we report that native neurotensin (NT1-13) or its C-terminal fragment, NT8-13 induced comparable increases in the amplitude of glutamatergic excitatory post-synaptic currents (EPSCs) in VTA neurons. The NT8-13 induced augmentation in Ih+ neurons was blocked by SR48692 (NTS1 antagonist) and SR142948A (NTS1/NTS2 antagonist), suggesting that the augmentation effect was mediated by NTS1 receptors. In Ih- neurons, however, only  SR142948A blocked the increase in the EPSC amplitude, indicating the involvement of NTS2.    In the second study we tested the effects of D-Tyr[11]NT (an active NT analog with differential binding affinities for NT receptor subtypes) on glutamatergic EPSCs in Ih+ and Ih- neurons and conducted (by Romrpe’s Lab) a parallel series of behavioral experiments using a conditioned place preference (CPP) paradigm. We found that D-Tyr[11]NT induced a dose dependent inhibition of EPSCs in Ih+ neurons that was mediated by the activation of NTS2 receptor. In contrast, D-Tyr[11]NT dose dependently enhanced glutamatergic EPSCs through an NTS1 receptor involvement in Ih- neurons. Results from behavioural experiments show that bilateral VTA microinjections of D-Tyr[11]NT induced a CPP that was blocked only by co-injection of SR142948A and SR48692, indicating a role for NTS1. This study allowed us to conclude that i) NT acts on VTA NTS1 receptors to induce a rewarding effect and ii) that this effect is due, at least in part, to an enhancement of glutamatergic inputs to non-dopamine (Ih-) neurons.  The third study entailed investigating the effects of D-Tyr[11]NT on isolated n-methyl-D-aspartate (NMDA) and  α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor mediated EPSCs in VTA neurons. We found that in Ih+  neurons both NMDA and AMPA EPSC amplitudes were attenuated by D-Tyr[11]NT. This attenuation appeared to be mediated by both NTS1 and NTS2 receptors. In Contrast, in Ih- neurons both NMDA and AMPA EPSC amplitudes were enhanced by an NTS1 dependent mechanism. Additionally, the enhancement effect resulted from a presynaptic potentiation of glutamatergic inputs. These results provide additional evidence that NT exerts a bidirectional modulation on glutamatergic neurotransmission in VTA neurons and highlights a novel peptidergic modulation of non-DA neurons that might be implicated in sensitization mechanisms.  Altogether, our studies allowed us to conclude that the NTergic modulation of glutamatergic neurotransmission in VTA DA and non-DA neurons is oppositely regulated by NTS2 and NTS1 receptors respectively. Additionally it highlights a peptidergic modulation of glutamatergic inputs to VTA non-DA neurons that might be crucial for addiction mechanisms.

Keyphrases: Dopamine, Glutamate, Patch-clamp, VTA, NT, EPSCs