Alpha-2 noradrenergic receptor activation inhibits the hyperpolarization-activated cation current (Ih) in neurons of the ventral tegmental area

Abstract

The ventral tegmental area (VTA) is the source of dopaminergic projections innervating cortical structures and ventral forebrain. Dysfunction of this mesocorticolimbic system is critically involved in psychiatric disorders such as addiction and schizophrenia. Changes in VTA dopamine (DA) neuronal activity can alter neurotransmitter release at target regions which modify information processing in the reward circuit. Here we studied the effect of alpha-2 noradrenergic receptor activation on the hyperpolarization-activated cation current (I(h)) in DA neurons of the rat VTA. Brain slice preparations using whole-cell current and voltage-clamp techniques were employed. Clonidine and UK14304 (alpha-2 receptor selective agonists) were found to decrease I(h) amplitude and to slow its rate of activation indicating a negative shift in the current's voltage dependence. Two non-subtype-selective alpha-2 receptor antagonists, yohimbine and RS79948, prevented the effects of alpha-2 receptor activation. RX821002, a noradrenergic antagonist specific for alpha-2A and alpha-2D did not prevent I(h) inhibition. This result suggests that clonidine might be acting via an alpha-2C subtype since this receptor is the most abundant variant in the VTA. Analysis of a second messenger system associated with the alpha-2 receptor revealed that I(h) inhibition is independent of cyclic AMP (cAMP) and resulted from the activation of protein kinase C. It is suggested that the alpha-2 mediated hyperpolarizing shift in I(h) voltage dependence can facilitate the transition from pacemaker firing to afferent-driven burst activity. This transition may play a key role on the changes in synaptic plasticity that occurs in the mesocorticolimbic system under pathological conditions.

Fig. 1. Clonidine’s effect on VTA DA cells

A. A 20 seconds bath application of the α-2 noradrenergic agonist clonidine (40 μM) evokes a slight depolarization of the membrane potential in VTA DA cells (1.7 ± 0.4 mV, n = 10). Concomitant with this event there is a significant decrease in the following measurements: action potential amplitude, afterhyperpolarization amplitude and, rate of subthreshold depolarization. There is a significant increase in the interspike interval. B. Current-clamp response of a VTA DA cell to a family of 8 hyperpolarizing dc current pulses of 100 pA each (duration: 1 sec, frequency: 1/9 Hz) in the absence and presence of clonidine. The cell was held at 0 pA. Note that all the effects seen above (part A of the figure) are also seen here. The amplitude of voltage responses under clonidine is increased indicating an augmentation in the cell’s input resistance. Voltage “sag” is also more profound and visibly slowed indicating a shift in the voltage dependence of the underlying I_h. C. In voltage clamp mode, bath application of clonidine reduces the difference between the steady state current (I_ss) and the instantaneous current (I_inst) at all command potentials below the holding level (−55 mV), i.e., clonidine inhibits I_h = I_ss − I_inst. In the presence of clonidine the membrane current reaches its steady state value at a much slower rate. As indicated by part B of the figure, clonidine’s effects on I_h, membrane potential and firing frequency follow a similar time course.

Fig. 2. Clonidine causes significant negative shift in I_h voltage dependence and Kir channels are not involved in this effect

A. Monoexponential time constants (τ) vs membrane potential plot indicates that clonidine (40μM) slows down I_h activation upon membrane hyperpolarization, i.e., greater τ. B. Normalized current-voltage curve showing that barium (Ba⁺²) does not alter clonidine’s effect on I_h, implying that Kir channels are not involved. C. Dose-response curve of clonidine’s reduction of I_h current amplitude at −125 mV, illustrating that there is an increase inhibition at higher concentrations. D. Activation curve showing that clonidine (40μM) produces a negative shift (−11.7 mV) in I_h voltage dependence (n = 12 is the same in both groups).

Fig. 3. Clonidine inhibits I_h by activation of the α-2 noradrenergic receptor

A. Application of the α-2 noradrenergic agonist UK14304 (20 μM) causes I_h inhibition in VTA DA cells. This inhibition is similar to the one evoked by 20 μM of clonidine (P > 0.05). The numbers above the bars indicate the number of cells that were used for each treatment. B. Current traces show that yohimbine, a selective non-subtype α-2 noradrenergic antagonist, blocks clonidine’s inhibitory effect. To the right, bar graphs summarize the effects of yohimbine. RS79948, another selective non-subtype- α-2 noradrenergic antagonist, also blocks clonidine’s action. Moxonidine (Mox) in the presence of yohimbine activates preferentially imidazoline receptors. The I_h was not decreased under this condition or under moxonidine alone. It seems that clonidine’s action is mediated by activation of α-2 receptors and do not involve an imidazoline receptor component. RX821002 (10 μM), an antagonist for α-2A and α-2D subtypes did not abolish clonidine’s evoked I_h inhibition. This inhibition was statistically different from that of RX 821002 alone (P < 0.05). Consequently, it appears that clonidine’s action is mediated by a specific α-2C receptor subtype. In all graphs the numbers above the bars indicate the number of cells that were used for each treatment.

Fig. 4. Activation of α-2 noradrenergic receptor stimulates PKC in a calcium insensitive manner

A. The bar graph shows that loading the patch pipette with 3 mM of cAMP does not block clonidine’s effect on I_h current. Minutes within the bar indicate the exposition time to cAMP. Note that there is not a significant difference between clonidine alone and clonidine plus cAMP (P > 0.05). The numbers above the bars indicate the number of cells that were used for each treatment. Asterisk denotes P < 0.05. B. The bar graph shows that bath application of 5 μM of the PKC activator phorbol 12,13 diacetate (PDA) inhibits I_h and occludes clonidine’s effect. Additionally, bath application of 1 μM of the PKC antagonist chelerythrine blocks clonidine’s action indicating that PKC is necessary for clonidine-induced Ih inhibition in VTA DA cells. Note that there is not a significant difference between clonidine alone and clonidine plus PDA (P > 0.05). The numbers above the bars indicate the number of cells that were used for each treatment. C. The bar graph shows that loading the patch pipette with 10 mM of the Ca²⁺ chelator BAPTA increases I_h amplitude. Minutes within the bar indicate the exposition time to BAPTA. This treatment does not block clonidine’s effect on I_h. Note that there is not a significant difference between clonidine alone and clonidine plus BAPTA (P > 0.05). The numbers above the bars indicate the number of cells that were used for each treatment. Asterisk denotes P < 0.05.