Information on TH gene regulation stems mainly from studies performed in models derived from epinephrine- or norepinephrine-secreting cells, like AM, sympathetic ganglia and LC. These studies have shown that TH is induced after prolonged stress, treatment with catecholamine-depleting drugs, like reserpine, or chronic treatment with cholinergic agonists (for reviews see (Kumer and Vrana 1996
; Sabban and Kvetnansky 2001
; Wong and Tank 2007
)). In AM and LC, these stimuli activate TH gene transcription rate and at least in the adrenal the response is dependent on presynaptic input. These studies have led to the hypothesis that induction of TH protein occurs after transsynaptic depolarization of catecholaminergic neurons and is due to an initial activation of the TH gene, followed by an increase in TH mRNA and subsequently an increase in TH protein. Other studies have provided evidence that post-transcriptional mechanisms and humoral factors may also participate in these responses.
There is much less information on TH gene regulation in midbrain dopamine neurons. Early reports suggested that many stressors that induce TH in AM, sympathetic ganglia and LC do not increase TH expression in midbrain. However, more recent reports have shown that some stressors elicit small increases in midbrain TH mRNA, but this response is highly dependent on the strain of the animal and it is not clear whether these increases lead to induction of TH protein (Sabban and Kvetnansky 2001
; Wong and Tank 2007
). Treatment of rats with the catecholamine-depleting drug, reserpine, leads to induction of TH mRNA in VTA neurons, but not SN neurons, suggesting that TH regulation differs in these two midbrain regions (Pasinetti et al. 1990
; Ortiz et al. 1996
). Even more puzzling is the finding that midbrain TH mRNA and TH gene transcription are not robustly induced by neurotoxins that deplete striatal dopamine, such as MPTP, 6-hydroxydopamine and ampthetamines (Pasinetti et al. 1992
; Sherman and Moody 1995
; Bowyer et al. 1998
; Rothblat et al. 2001
). In the periphery, depletion of norepinephrine from sympathetic nerve terminals leads to relatively large compensatory increases in TH mRNA and TH protein in sympathetic ganglia and AM (Kumer and Vrana 1996
). Why this type of compensatory response does not occur in nigrostriatal neurons is not clear. It is possible that appropriate signals are not transmitted to midbrain dopamine neurons to elicit a compensatory induction of TH mRNA when striatal nerve terminals are destroyed. However, it is difficult to test this hypothesis, because so little is known about the signaling mechanisms that control midbrain TH expression in response to extracellular stimuli. This lack of knowledge is unfortunate, because similar findings (small or insignificant changes in TH mRNA in surviving midbrain dopamine neurons) are observed in autopsy samples from Parkinsonian patients (Javoy-Agid et al. 1990
; Kastner et al. 1993
). Furthermore, it is possible that inappropriate regulation of TH gene expression may play a role in drug addiction or schizophrenia, diseases that are mediated by VTA neurons.
Nicotine stimulates TH gene transcription, but does not induce TH mRNA or TH protein in cultured midbrain model systems
Our results show that nicotine stimulates TH gene transcription in a dose-dependent manner in both midbrain explant slice cultures and MN9D cells. Nicotine elicits a 2–4 fold stimulation of the TH gene and this response is totally blocked by the nAChR antagonist MLA. This latter finding is in agreement with the results of Serova and Sabban (2002)
, who demonstrate that MLA blocks the induction of midbrain TH mRNA by nicotine under in vivo conditions. At the concentration used in our studies (1 µ M), MLA effectively blocks nAChRs, but not muscarinic AChRs or other types of receptors; however, it is not selective for alpha-7 nAChRs at this concentration (Davies et al. 1999
). We did not perform a detailed pharmacological analysis of the nAChRs responsible for this transcriptional effect, because it does not lead to enhanced TH expression. Nevertheless, the results are consistent with a model in which nicotine acts directly on nAChRs in midbrain to stimulate TH gene transcription. Furthermore, the results using the MN9D cells argue that at least part of this response may be mediated by nAChRs present on dopamine neurons.
A second important finding is that in both culture model systems transcriptional activation by nicotine is relatively transient, lasting for at least 1 hr, but less than 3 hr. No increases in transcription are observed at later time points. The kinetics of this transcriptional activation has important consequences. Nicotine activates TH gene transcription for a relatively brief period of time, and this transient activation does not lead to significant increases in TH mRNA or protein. This latter finding is consistent with results from previous studies investigating the effect of nicotine on TH gene expression in rat AM (Fossom et al. 1991
). When nicotine is administered once to rats, adrenal TH gene transcription is activated 2–3 fold for up to 1 hr, but less than 3 hr. This activation is not associated with TH mRNA induction. In contrast, when nicotine is administered repeatedly to rats over a three hr period, TH gene transcription is activated for 3–6 hr and this leads to induction of TH mRNA and TH protein. These results suggest that TH gene transcription must be increased for a period of time greater than 1–3 hr in order to produce enough TH mRNA to measure its accumulation. The results in the midbrain models are in agreement with this hypothesis. In contrast, these midbrain results differ from those observed in PC12 cells treated with nicotine (Gueorguiev et al. 2000
). In PC12 cells nicotine elicits a sustained increase in intracellular calcium, which leads to a sustained induction of TH mRNA. The reason for the disparate results between PC12 cells and midbrain models remains unclear. It is possible that midbrain nAChRs are desensitized after sustained exposure to nicotine. Alternatively, nicotine may activate different downstream signaling pathways and/or transcription factors. More work is needed to clarify this issue.
Nicotine-mediated induction of TH mRNA in midbrain in rats is dependent on the GR
Under in vivo conditions, nicotine may be acting by multiple mechanisms to stimulate TH gene transcription in midbrain neurons. It may be interacting directly with nAChRs present on dopamine neurons or on presynaptic nerve terminals of afferent neurons that innervate dopamine cell bodies or dendrites. Nicotine may also be acting at other sites in the brain, activating stimulatory input or disinhibiting inhibitory input into the midbrain. All of these possibilities may activate signaling pathways in dopamine neurons, leading to stimulation of the TH gene. Another possible mechanism by which nicotine may act in vivo is via elevation of humoral factors that act on dopamine neurons to induce TH. We have tested the hypothesis that glucocorticoids may participate as a humoral factor in this response.
It is also well-established that glucocorticoids induce TH gene expression in cultured neuroblastoma and pheochromocytoma cells (Tank and Weiner 1982
; Fossom et al. 1992
). However, it is not clear whether glucocorticoids induce TH in other model systems and the in vivo response to glucocorticoids remains ambiguous. Early evidence showed that glucocorticoids induce TH in sympathetic ganglia (Otten and Thoenen 1975
). However, subsequent studies have contradicted these findings. Sze and Hendrick (1983)
have shown that glucocorticoids other than dexamethasone do not induce TH in sympathetic ganglia and that dexamethasone may be working by stimulating release of acetylcholine from preganglionic neurons. Sabban and coworkers (Nankova et al. 1996
; Serova et al. 2008
) have demonstrated that administration of cortisol to adrenalectomized rats does not induce TH in sympathetic ganglia and have presented evidence that the ganglionic response to stress is due to the direct action of ACTH, not glucocorticoids.
The role of glucocorticoids in regulating TH gene expression in brain is also unclear. An early report suggested that glucocorticoids induce TH in LC (Markey et al. 1982
). However, subsequent studies have concluded that glucocorticoids do not induce TH mRNA in LC, nor do they play an important role in the response of LC to stress (Markey and Sze 1984
; Makino et al. 2002
). In regard to midbrain, McArthur et al (2007)
have shown that perinatal exposure to dexamethasone in the drinking water alters the size and morphology of midbrain dopamine neurons into adulthood. Furthermore, it has been shown that glucocorticoids increase stimulated-dopamine release, alter dopamine turnover in midbrain neurons and modify dopamine-induced behaviors (Slotkin et al. 1992
; Piazza et al. 1996
; Rouge-Pont et al. 1999
). However, to our knowledge, the present study is the first to directly demonstrate that glucocorticoids induce TH gene expression in cell model systems derived at least partially from midbrain origins. Our results clearly establish that the synthetic glucocorticoid dexamethasone stimulates TH gene transcription rate and induces TH mRNA and TH protein in both midbrain slice cultures and MN9D cells. The transcriptional response is sustained for at least 24 hr. The fact that mifepristone blocks these responses are consistent with it being mediated by GRs present in the MN9D cells. These results support the hypothesis that glucocorticoids are capable of inducing TH gene expression in midbrain and consequently may participate in the nicotine-mediated response.
A major finding in the present study is that the induction of midbrain TH mRNA by nicotine under in vivo conditions is inhibited by mifepristone. The nicotine response in the SN is totally blocked, whereas that in the VTA is inhibited by 50–60% by the GR antagonist. These results support the idea that under in vivo conditions the nicotine-mediated induction of midbrain TH mRNA is dependent at least partially on GRs. A number of methodological and conceptual considerations need to be discussed. First, the dose of mifepristone (10–20 mg/kg per day) used in these experiments has been shown to inhibit GR-mediated central nervous system effects in numerous reports (Joels et al. 2003
; Yang et al. 2004
; Wong and Herbert 2005
; Avital et al. 2006
; Dong et al. 2006
). Furthermore, doses as high as 25 mg/kg have been shown to inhibit GR in brain without inhibiting the mineralocorticoid receptor (MR) (Wong and Herbert 2005
; Avital et al. 2006
). Secondly, we chose to administer the drug 15 min prior to each nicotine injection, because it has a relatively short half-life (~6–8 hr) in rats (He et al. 2007
). Thirdly, GRs have been colocalized with TH in midbrain neurons (Harfstrand et al. 1986
); consequently, it is feasible that glucocorticoids may act directly on these GRs in dopamine neurons to produce their effects on TH gene transcription. Finally, mifepristone is a highly specific antagonist of GRs, progesterone receptors and androgen receptors (Lu et al. 2006
); consequently, it may be blocking all three of these receptors in our in vivo studies. However, to our knowledge, there is no evidence that nicotine elevates circulating levels of either progesterone or testosterone in male rats. Hence, even though it is possible that progesterone and/or androgen receptors may play a role in regulating TH gene expression under basal conditions or during development, it seems unlikely that these receptors mediate the observed response to nicotine in midbrain. It is also possible that MRs may participate in the nicotine response, since these receptors have high affinity for corticosterone (de Kloet et al. 2008
). Mifepristone does not bind to MRs with high affinity (Lu et al. 2006
); however, the drug is a substrate for the P-glycoprotein extrusion pump in the blood-brain barrier (de Kloet et al. 2008
). Hence, it is difficult to predict what the concentration of mifepristone is at its site of action in brain and at high concentrations it is possible that MRs are blocked (Tanaka et al. 1997
; de Kloet et al. 2008
). MRs are primarily localized in limbic structures and prefrontal cortex (de Kloet et al. 2008
) and to our knowledge, have not been definitively localized in midbrain dopamine neurons. Nevertheless, the involvement of mineralocorticoid receptors in TH gene regulation has not been investigated and our results do not rule out their potential participation in this response.
Taken together, one explanation for our results is that nicotine elevates circulating glucocorticoids, which pass into the brain and directly induce TH gene expression in midbrain dopamine neurons by interaction with GRs in these neurons and the consequent stimulation of TH gene transcription rate. This hypothesis is consistent with the results obtained using the cultured slice and MN9D cell model systems and the fact that midbrain dopamine neurons are known to express GR receptors (Harfstrand et al. 1986
; Ronken et al. 1994
). This hypothesis would also explain the discrepancy between the in vivo results, in which nicotine induces midbrain TH mRNA, and the cell culture results, in which nicotine does not induce TH mRNA in midbrain slices or MN9D cells. However, there are other models that also explain the available data. Glucocorticoids may be acting on afferent neurons or nearby glial cells to stimulate the synthesis and release of other factors that induce TH in midbrain dopamine neurons. Alternatively, glucocorticoids may be having a permissive effect, inducing factors in dopamine neurons, which are necessary for TH induction by other unknown extracellular stimuli. For instance, glucocorticoids may induce intracellular factors that respond to the activation of nAChRs by nicotine. On the other hand, glucocorticoids may be acting peripherally to elevate an unknown humoral factor, which then passes into brain and produces the response in midbrain neurons. These different possibilities need to be investigated. However, our findings suggest that nicotine’s effects on midbrain TH gene expression are not solely due to the actions of nicotine on midbrain nAChRs, but require the elevation of glucocorticoids and subsequent activation of GRs.
Understanding nicotine’s effects on TH gene expression in midbrain dopamine neurons is important in a number of contexts. Nicotine’s addictive properties are at least partially mediated by its ability to stimulate dopamine release from mesocorticolimbic neurons. Furthermore, it is well-established that smokers have a lower incidence of Parkinson’s disease, possibly because the nicotine in cigarettes promotes dopamine release from nigrostriatal neurons. In both these contexts, it would be expected that chronic exposure to nicotine would induce TH gene expression as an adaptive mechanism, to maintain dopamine levels in these neurons. Hence, an understanding of the mechanisms that regulate TH expression in these midbrain neurons in response to nicotine may be useful for treating these disorders. Furthermore, schizophrenia is associated with excessive release of dopamine from mesocorticolimbic neurons and Parkinson’s disease is due to the loss of nigrostriatal neurons. Since very little is known about the mechanisms that control TH gene expression in midbrain dopamine neurons, the results of this study may help to provide new therapeutic avenues to either down-regulate or up-regulate TH expression and hence dopamine biosynthesis during these diseases.