Chronic morphine alters VTA DA neuron morphology, activity, and output to NAc
We set out to characterize the effects of chronic morphine on several phenotypic characteristics of VTA DA neurons. We first determined whether morphine induces a morphological change in the mouse VTA similar to that seen in rats. We found a ~25% decrease in the mean surface area of mouse VTA DA neurons in response to chronic morphine (), very similar to the magnitude of soma size decrease observed in rats (Russo et al., 2007
; Sklair-Tavron et al., 1996
). To study the clinical relevance of this finding, we examined postmortem sections of human brain and observed a significant decrease in the soma size of VTA DA neurons in heroin users compared to controls ().
Morphine Alters VTA DA Neuronal Morphology, Activity, and Output to NAc
We next examined whether the chronic opiate-induced morphological change was correlated with changes in DA neuronal excitability. We found that chronic morphine-treated mice, compared to sham-treated mice, exhibited an increase in the spontaneous firing rate of VTA DA neurons in brain slices (). This effect was not dependent on residual morphine in the slice, since blockade of opioid receptors with naloxone did not affect cell excitability (). Moreover, the inclusion of a low dose of morphine (5 μM) in the bath solution to prevent “withdrawal” in the slice did not alter DA neuron firing rate ().
Given the observations that chronic morphine decreases the size of VTA DA neurons, but concomitantly increases their excitability, it was important to determine whether net DA output from VTA is altered. We examined levels of extracellular DA in nucleus accumbens (NAc) in vivo
, widely considered a key determinant of reward (Hyman et al., 2006
). In opposition to the increased firing rate, we found that chronic morphine dramatically decreased electrically-evoked DA output in NAc of rats as measured by fast-scan cyclic voltammetry (). This reduction in DA output from VTA DA neurons supports the notion that the reduced soma size of the neurons, induced by chronic morphine, correlates with functional output, consistent with the reward tolerance induced by chronic morphine under these conditions (Russo et al., 2007
Alteration of VTA neuronal activity regulates DA soma size
Next, we examined a possible relationship between the increase in VTA DA neuron firing rate and soma size decrease, with the hypothesis that the increased firing rate per se
induces changes in soma size. We virally overexpressed a dominant-negative K+
channel subunit (dnK, KCNAB2-S188A, R189L) locally within VTA; we showed previously that this mutant channel increases the firing rate of VTA DA neurons (Krishnan et al., 2007
). Overexpression of dnK was sufficient to decrease the surface area of VTA DA neurons (). To obtain the converse type of information, we virally overexpressed wild-type Kir2.1 in VTA, which we showed decreases DA neuron firing rate (Krishnan et al., 2007
). While overexpression of Kir2.1 alone did not affect VTA DA soma size (data not shown), it completely blocked the ability of chronic morphine both to decrease soma size () and to increase DA neuron firing rate (). These findings support our hypothesis that the morphine-induced increase in VTA DA neuron excitability is both necessary and sufficient for mediating the decrease in soma size.
Alteration of VTA DA Firing Rate is Necessary and Sufficient for Morphine-Induced Changes in Soma Size
Chronic morphine alters K+ channel expression in VTA
Given the increase in VTA DA neuronal firing rate observed in response to chronic morphine, we examined possible underlying mechanisms. One possibility is that morphine, by downregulating AKT activity in these neurons (Russo et al., 2007
), might increase GABAA
currents, as demonstrated previously (Krishnan et al., 2008
). Another possibility is that K+
channels may be modulated, since many conditions associated with altered neuronal excitability involve changes in K+
channel expression (Luscher and Slesinger, 2010
). To test this possibility, we examined VTA from mice that received sham or morphine pellets and analyzed by PCR the expression of K+
channels whose regulation has been implicated in other systems. We observed a significant decrease in expression levels of two K+
channel subunits, KCNAB2
, with a trend seen for several others ().
Chronic Morphine Decreases the Expression of K+ Channel Subunits in VTA
To examine whether decreased K+ channel expression is regulated at the transcriptional level, we performed chromatin immunoprecipitation (ChIP) on VTA dissected from sham- and morphine-treated rats. Overall, we observed epigenetic changes consistent with K+ channel downregulation (). Modifications consistent with increased transcription—acetylation of histones H3 and H4, trimethylation of Lys-4 of H3, and binding of RNA polymerase II (POL2)—were significantly decreased at the KCNAB2 and KCNF1 promoters () and decreased trimethylation of Lys-4 of H3 and binding of RNA polymerase II (POL2) was observed at the GIRK3 promoter. These changes suggest that, in addition to morphine potentially reducing GABAA responses in VTA DA neurons, expression of specific K+ channel subunits is reduced via transcriptional mechanisms to further mediate enhanced excitability of these neurons.
Since both chronic morphine and decreased AKT signaling increase VTA DA neuron firing rate, we next examined whether decreasing AKT activity also decreases K+ channel expression similarly to chronic morphine. To test this possibility, we analyzed VTA from mice that had received intra-VTA injections of either HSV-GFP or HSV-IRS2dn. We overexpressed IRS2dn because this is the most direct way of reducing AKT activity without affecting total levels of the enzyme, as seen with chronic morphine. We observed a significant decrease in levels of expression of three K+ channel subunits, KCNF1, KCNJ2, and GIRK3 (). These data suggest that, in addition to altering DA neuronal activity via GABAA channel regulation, altering AKT signaling can also modulate K+ channel expression.
VTA DA Neuron Firing Rate, Cell Morphology, and Output to NAc are Functionally Linked
Finally, since both chronic morphine and decreased IRS2/AKT signaling control VTA DA neuronal morphology and excitability, we determined whether decreased AKT signaling also affects DA output to NAc. As found with chronic morphine, HSV-IRS2dn in VTA decreased electrically-evoked DA output in rat NAc ().
Based on our prior research in rats showing that IRS2 downregulation mediates the chronic morphine-induced decrease in VTA DA soma size (Russo et al., 2007
), we assumed that this morphological change was likewise dependent on AKT downregulation. To verify this directly, we virally overexpressed either a dominant-negative (AKTdn) or constitutively-active (AKTca) AKT mutant and examined VTA DA neuron morphology. As predicted, AKTdn significantly decreased VTA soma size, while AKTca increased it (). Together, these data establish that chronic morphine and reduced IRS2/AKT signaling decrease VTA DA soma size, K+
channel expression, and DA output to NAc.
Chronic morphine alters mTOR signaling in VTA
To investigate other signaling changes associated with IRS2/AKT that mediate morphological and physiological adaptations to opiates, we first collected VTA from chronic morphine-treated rats. Consistent with previous observations (Russo et al., 2007
; Wolf et al., 1999
), we found a morphine-induced decrease in levels of total IRS2 and of phospho-AKT, with no change in total levels of AKT, in VTA (Figure S1A
). The decrease in phospho-AKT was associated, as would be expected, with a decrease in levels of phospho-glycogen synthase kinase-3β (GSK3β), a major downstream target of AKT, with no change in total GSK3β levels. Another indirect downstream target of AKT is mTORC1. However, in contrast to an expected decrease in mTORC1 activity, we obtained evidence for increased mTORC1 signaling in VTA in response to chronic morphine, based on increased levels of phospho-S6 and phospho-4EBP (Figure S1A
). Total levels of S6 and 4EBP were also increased, as was the positive regulator of mTORC1 activity, ras homolog enriched in brain (Rheb). There was also a non-significant trend for increased phosphomTOR and phospho-p70S6K, a direct target of mTORC1. These data indicate that morphine increases mTORC1 signaling in VTA coincident with a decrease in upstream IRS2/AKT signaling.
We determined whether these changes in IRS2/AKT and mTORC1 signaling were specific to VTA or were a generalized response to chronic opiates. In contrast to VTA, we did not observe any opiate-induced changes in the IRS2/AKT/mTORC1 signaling pathway in NAc (Figure S1B
), suggesting some specificity of opiate-induced adaptations within the brain reward circuit.
We next examined whether chronic morphine produced comparable biochemical changes in mice. We found, analogous to data from rats, that levels of IRS2 and phospho-AKT (Thr-308) were decreased in mouse VTA by chronic morphine, while levels of phospho-S6 were increased (). Total levels of AKT and S6 were unaffected. We also observed increased levels of phospho-p70S6K. In contrast, we observed no morphine regulation of proteins immediately upstream of mTORC1, including Rheb, TSC1, or TSC2. Note that we were unable to assess levels of phospho- and total 4EBP in mouse VTA, since available antibodies did not yield a reliable signal.
Morphine Regulation of mTORC1 and mTORC2 Activity in VTA
Since mTOR forms two distinct signaling complexes, mTORC1 and mTORC2, we investigated whether activity of both complexes was altered by chronic morphine. While the Thr-308 site of AKT is phosphorylated by PDK, the Ser-473 site is a substrate of mTORC2, and phosphorylation of both sites is associated with increased AKT catalytic activity (Sarbassov et al., 2005b
). As with the Thr-308 site, we observed a morphine-induced decrease in levels of phospho-AKT at Ser-473 in VTA (). Chronic morphine also decreased the phosphorylation state of another target of mTORC2, PKCα, in this brain region. We did not detect any changes in levels of phospho- or total mTOR, nor changes in its associated proteins, Raptor or Rictor. Together, these data show that chronic morphine decreases AKT signaling in VTA, which is associated with an increase in mTORC1 signaling but a decrease in mTORC2 signaling. Importantly, we did not observe any changes in IRS2/AKT, mTORC1, or mTORC2 signaling in VTA of mice that overexpressed dnK (Figure S1C
), suggesting that increased VTA neuronal activity per se
is not sufficient to induce changes in these signaling pathways. While it is well established that IRS2/AKT signaling is an upstream mediator of mTORC1 activity, regulation of mTORC2 activity is not well defined. It has been suggested that decreased growth factor signaling may decrease mTORC2 activity through reduced phosphatidylinositol-3-kinase (PI3K) activity (which is downstream of IRS2) (Foster and Fingar, 2010
; Oh and Jacinto, 2011
). In support of this possibility, we found that IRS2dn overexpression in cultured pheochromocytoma cells decreases phospho-AKT at its mTORC2 (Ser-473) site (Russo et al., 2007
). When we overexpresssed IRS2dn in mouse VTA, we observed the expected decrease in phospho-AKT Thr-308 (GFP:100.0±8.8% n=5, IRS2dn:65.7±7.6% n=8, t-test, p<0.05), plus a trend for decreased phospho-AKT Ser-473 (GFP:100.0±7.5 n=5, IRS2dn:68.8±11.4% n=8, t-test, p=0.07), suggesting that this regulation may also occur in VTA in vivo
Since the increase in mTORC1 signaling was unexpected given the decreases in phospho-AKT and VTA DA soma size, we determined whether induction of mTORC1 activity was occurring within VTA DA neurons. We performed immunohistochemistry on VTA sections taken from morphine- or sham-treated mice and found increased colocalization of phospho-S6 and tyrosine hydroxylase (TH), a marker of DA neurons, in response to chronic morphine (). The specificity of the phospho-S6 signal was validated by rapamycin (a selective inhibitor of mTORC1): sections from mice treated with rapamycin (30 mg/kg, i.p. daily, 6 days) showed no detectable phospho-S6 signal within VTA (). Further, the morphine-induced increase in phospho-S6+ cells was specific for TH+ cells within VTA, as there was no evidence for an increase in the number of phospho-S6+, TH– cells (sham: 2.39±0.69 cells/scan, morphine: 1.5±0.31 cells/scan, N=18 mice, p>0.1). However, there was no difference in mean soma size of TH+ DA neurons that were either phospho-S6+ or – (), showing that phospho-S6 status was not correlated to DA soma size.
We next used rapamycin to directly assess whether the increase in mTORC1 activity was integral to the morphine-induced morphology changes. We administered rapamycin (10 or 30 mg/kg i.p. daily for 6 days) to mice and found the expected dose-dependent decrease in levels of phospho-mTOR and phospho-S6 in VTA (). We did not observe a significant decrease in phospho-p70S6K, but this may be due to an increase in total p70S6K levels induced by this treatment. This effect of rapamycin was specific for mTORC1, since there was no evidence of altered mTORC2 activity, based on normal levels of phospho-AKT (Ser-473) and phospho-PKCα, in VTA. Rapamycin treatment of morphine-naïve mice had no effect on VTA DA cell surface area, demonstrating that decreasing mTORC1 activity per se is not sufficient to alter the size of these neurons (). Further, when mice were pretreated with rapamycin and then treated chronically with morphine, we still observed the expected morphine-induced decrease in DA soma size. These findings show that preventing the morphine-induced increase in mTORC1 signaling in VTA does not block the morphine-induced decrease in soma size.
mTORC2 activity regulates morphine-induced changes in cell morphology, cell physiology, and behavior
Since there is no selective small molecule inhibitor of mTORC2, we used a conditional neuronal knockout strategy, with recently developed floxed-Rictor mice (Siuta et al., 2010
) to directly study the contribution of mTORC2 in morphine action. Knocking out Rictor enables a selective reduction in mTORC2 activity, without any discernable effect on mTORC1. To achieve a local knock-out of Rictor from VTA, we injected AAV-Cre into VTA of floxed-Rictor mice or into wild-type littermates as a control (). Knock-out was validated by RT-PCR and western blot analysis, where we observed a significant decrease in Rictor mRNA in VTA and decreased phosphorylation of the mTORC2 substrates AKT (Ser-473) and PKCα (Figure S2A
). Local Rictor knock-out also decreased DA cell surface area by ~20% (). We next developed an HSV to overexpress Rictor-T1135A. This Rictor mutant increases mTORC2 activity, and lacks the p70S6K phosphorylation site, eliminating the possibility of mTORC1 negative feedback regulation of mTORC2 (see Discussion). This vector increased Rictor expression and mTORC2 signaling in VTA (Figure S2B
), and blocked the morphine-induced decrease in DA neuron soma size (). These results demonstrate that downregulation of mTORC2 signaling in VTA is both necessary and sufficient for mediating the morphine-induced decrease in DA soma size.
Downregulation of mTORC2 Activity Mediates Morphine Effects on VTA Morphology and Neuronal Activity and on Reward Behavior
In addition to the mTOR pathway, another downstream target of AKT that has been observed to affect neuronal size and structure in other systems is GSK3β (van Diepen et al., 2009
). Since we observe changes consistent with increased GSK3β activity (decreased phospho-GSK3β, Figure S1A
) in VTA after chronic morphine, we studied the possible influence of GSK3β in regulating VTA DA soma size. Overexpression of wild-type GSK3β in VTA, which mimics morphine regulation of the protein, did not alter soma size (Figure S3
). Additionally, when we overexpressed a dominant-negative mutant of GSK3β (K85A-K86A) to block the morphine-induced increase in enzyme activity, we still observed the expected decrease in soma size. These data suggest that GSK3β activity in VTA is not involved in morphine-induced changes in the morphology of VTA DA neurons.
To investigate whether mTORC2 downregulation might also contribute to the morphine-induced increase in DA neuron firing rate, we injected HSV-Rictor-T1135A into mouse VTA, treated the mice with sham or morphine pellets, and recorded DA neuron firing rates in acute VTA slices. Similarly to , chronic morphine increased DA neuron firing rate in both GFP-positive and GFP-negative cells compared to sham-treated mice (). However, in cells that overexpressed Rictor, the morphine-induced increase in firing rate was completely abolished (). Further, it was the overexpression of Rictor in the DA neurons themselves driving the effect, as GFP-negative DA neurons from the Rictor-morphine mice still showed the morphine-induced increase in firing rate. These data support a cell-autonomous link between decreased mTORC2 activity and increased VTA DA neuron excitability induced by chronic morphine.
Given that Rictor overexpression prevented morphine-induced changes in VTA neuron morphology and excitability, we next assessed whether altered mTORC2 activity might also affect morphine reward as measured by place conditioning. We found that Rictor overexpression caused a significant place preference to a low dose of morphine (5 mg/kg) that does not induce preference in GFP-injected mice (). Rictor overexpression also increased morphine-induced locomotor activity (). Conversely, local knock-out of Rictor in VTA decreased morphine place preference (15 mg/kg) without affecting locomotor activity (). These data are consistent with our previous findings that treatments that decrease VTA DA soma size—chronic morphine or decreased AKT signaling—decrease morphine reward.