Presynaptic inhibition of glutamate transmission by α2 receptors in the VTA

Abstract

The ventral tegmental area (VTA) forms part of the mesocorticolimbic system and plays a pivotal role in reward and reinforcing actions of drugs of abuse. Glutamate transmission within the VTA controls important aspects of goal-directed behavior and motivation. Noradrenergic receptors also present in the VTA have important functions in the modulation of neuronal activity. Here we studied the effects of α2 noradrenergic receptor activation in the alteration of glutamate neurotransmission in VTA dopaminergic neurons from male Sprague-Dawley rats. We used whole-cell patch-clamp recordings from putative VTA dopaminergic neurons and measured excitatory postsynaptic currents. Clonidine (40 μm) and UK 14,304 (40 μm), both α2 receptor agonists, reduced (approximately 40%) the amplitude of glutamate-induced excitatory postsynaptic currents. After clonidine administration, there was a dose-dependent reduction over the concentration range of 15-40 μm. Using yohimbine (20 μm) and two other α2 adrenergic receptor antagonists, idaxozan (40 μm) and atipemazole (20 μm), we demonstrated that the inhibitory action is specifically mediated by α2 receptors. Moreover, by inhibiting protein kinases with H-7 (75 μm), Rp-adenosine 3',5'-cyclic (11 μm) and chelerythrine (1 μm) it was shown that the clonidine-induced inhibition seems to involve a selective activation of the protein kinase C intracellular pathway. Increased paired-pulse ratios and changes in spontaneous and miniature excitatory postsynaptic current frequencies but not amplitudes indicated that the effect of the α2 agonist was presynaptically mediated. It is suggested that the suppression of glutamate excitatory inputs onto VTA dopaminergic neurons might be relevant in the regulation of reward and drug-seeking behaviors.

Fig. 1. Bath application of clonidine reduces AMPA EPSCs amplitude in putative VTA DA neurons

A. Representative recordings from the same cell illustrating that clonidine superfusion (40 μM), induces a significant reduction in AMPA EPSCs amplitude in a putative VTA DA cell voltage clamped at −70 mV. The effect was reversible since it returned to near control values after a washout period. B. Summary time course of the effect of clonidine bath application on AMPA EPSCs amplitude recorded from 10 putative VTA DA neurons. A 10 min clonidine (40 μM) application causes depression of AMPA EPSCs. There was a rapid return to control levels upon clonidine removal. All recordings were made at 5 minutes intervals. C. Bar graph showing that on average clonidine application resulted in a ~ 40% decrease on AMPA EPSCs amplitude. No significant differences were observed between control (99.4 ± 0.5 %) and washout period (94.8 ± 8.2 %). This effect was specific to AMPA EPSCs as GABAa IPSCs were not affected by clonidine treatment (control: 105 ± 4 % vs. clonidine 103 ± 7 %, p=0.82). D. The clonidine-induced inhibition was dose-dependent over the concentration range of 15 to 40 μM. Numbers in parenthesis indicate the sample size used to test each concentration. *** p= 6 × 10⁻⁵

Fig. 2. Alpha-2 antagonists block clonidine effect on AMPA EPSCs

A. Representative recordings from the same cell showing that the alpha2-antagonist yohimbine (20 μM) completely abolishes the clonidine-evoked inhibition of AMPA EPSCs. B. Summary time course illustrating that the alpha2-antagonist yohimbine (20 μM) blocks the clonidine-evoked inhibition of AMPA EPSCs. Note that yohimbine alone has no effect on EPSCs amplitude. At each plotted point n = 9. C. Superfusing alpha2-antagonists yohimbine (Yoh) or atipamezole (Atip) leaves AMPA EPSCs amplitude unaltered. D. Cosuperfusing clonidine (40 μM) plus yohimbine (20 μM) or atipamezole (20 μM) prevents the clonidine-evoked reduction of AMPA EPSCs amplitude.

Fig. 3. The alpha-2 receptor mediated decrease in AMPA EPSCs amplitude results from the activation of PKC signaling

A. Representative recordings from the same cell showing that chelerythrine (1 μM), a selective PKC inhibitor, completely prevents the clonidine-evoked inhibition of AMPA EPSCs in VTA DA cells. B. Bar graph demonstrating that superfusion of the non-specific protein kinase inhibitor H-7 (75 μM) blocked clonidine’s inhibitory effect (40 μM, 10 min application) on AMPA EPSCs amplitude in VTA DA cells. Note that H7 alone had no effect on AMPA EPSCs suggesting that inhibition of PKA signaling does not affect EPSCs amplitude. C. Bar graph showing that application of Chelerythrine (Chelery), a potent PKC inhibitor, completely abolished clonidine’s inhibitory action (40 μM, 10 min application) on AMPA EPSCs in VTA DA cells. Note that Chelerythrine alone had no effect. D. Rp, a specific PKA inhibitor did not block clonidine’s s effect. ** p= 0.009

Fig. 4. Clonidine bath application increases Paired-Pulse Ratio (PPR) in putative VTA DA neurons

A. Representative recordings from the same cell illustrating that clonidine superfusion (40 μM, 10 min application), induces a significant increase in paired pulse ratio (PPR = EPSC₂/EPSC₁) in a putative VTA DA neuron voltage clamp at −70 mV. Note that the time interval between consecutive EPSCs is 50 ms. B. Graph summarizing the change in PPR of 6 cells after a 10 min clonidine (40 μM) bath application. C. Bar graph showing that the clonidine-evoked increased in PPR is statically significant and specific to AMPA EPSCs PPR as no effect was evoked by a similar treatment onto GABAa IPSCs PPR. D. Clonidine’s effect on PPR is blocked by yohimbine an alpha-2 antagonist. ** p= 0.002

Fig. 5. Clonidine reduces sEPSCs frequency with no effect on its amplitude

A. Representative recordings from the same cell illustrating that clonidine application (40 μM) decreases sEPSCs frequency without affecting its amplitude. The cell was voltage clamped at −70 mV during the recordings. B. Clonidine superfusion (40 μM, 10 min. application) results in a positive shift of the inter-event interval cumulative distribution (K-S, p = 0.01) implying a decrease in sEPSCs frequency. The plot was constructed from a single cell recording distinct from cell in part A. C. Clonidine application does not shift the amplitude cumulative distribution (K-S, p = 0.43). Thus, clonidine has no effect on sEPSCs amplitudes. The plot was constructed from the cell used in B. D. Summary graph showing that clonidine superfusion decreases sEPSCs frequency without affecting its amplitude. *p=0.026

Fig. 6. Clonidine reduces mEPSCs frequency with no effect on its amplitude

A. Representative recordings from the same cell illustrating that in the presence of TTX (0.5 μM) clonidine application (40 μM) decreases mEPSCs frequency without affecting its amplitude. The cell was voltage clamped at −70 mV during the recordings. B. Clonidine superfusion (40 μM, 10 min. application) results in a positive shift of the inter-event interval cumulative distribution (K-S, p=0.0007) implying a decrease in mEPSCs frequency. The plot was constructed from a single cell recording distinct from cell in part A. C. Clonidine application does not shift the amplitude cumulative distribution (K-S, p=0.72). Thus, clonidine has no effect on mEPSCs amplitudes. The plot was constructed from the cell used in B. D. Summary graph showing that clonidine superfusion in the presence of TTX decreases mEPSCs frequency without affecting its amplitude. *p=0.011