In the current study we report that β-AR activation increased sEPSCs in the dlBNST, a component of the extended amygdala that receives a dense noradrenergic projection and is activated by both stressors and drugs of abuse (42
). These effects were mediated through β1
-ARs and required CRFR1 signaling. Our earlier studies indicated that NE-induced increases in evoked field potentials are dependent upon β2
-AR activation (29
), suggesting that multiple β-AR subtypes participate in regulation of excitatory transmission in the dlBNST. In parallel, differing roles of BNST β-AR subtypes have been proposed at the behavioral level. In animals highly reactive to morphine withdrawal, a β1
-AR antagonist injected into the BNST blocks withdrawal induced conditioned place aversion (30
). On the other hand, a β2
-AR antagonist injected into the BNST dose-dependently attenuated intraplantar-formalin-induced CPA (48
We previously found that DA, through multiple DA receptors, also increases sEPSC frequency in a CRFR1-dependent manner in the dlBNST (25
). Interestingly, while both the actions of DA receptors and β-ARs on excitatory transmission in BNST required CRFR1 signaling, the actions of DA were specific to sEPSCs, while β-AR activation modulates both sEPSCs and evoked EPSCs. In addition, we found that DA could still produce a further enhancement of sEPSCs under a period of maximal β-AR activation and vice versa. Thus these data suggest the interesting possibility that DA and NE may modulate distinct populations of excitatory synapses in the BNST.
Finally, we found that CRFR1 dependent modulation of excitatory transmission in the dlBNST is disrupted by repeated cocaine administration. The CRFR1 actions return after 10 days of withdrawal, but now appear to be more labile, as they are disrupted by a single cocaine challenge.
β-AR activation enhances BNST glutamate release in a CRFR1-dependent manner
The BNST receives both dopaminergic projections (49
) as well as a dense noradrenergic input from the nucleus tractus solitarius via the ventral noradrenergic bundle (51
). Our previous and current results indicate that both DA and NE can enhance glutamatergic transmission in the dlBNST. We speculate that in both cases the CRFR1s that mediate this increased glutamate release are localized on presynaptic glutamatergic terminals in the dlBNST. Indeed, immunohistochemical evidence suggests that CRFR1 is expressed on excitatory terminals in BNST(52
The BNST is an important region for the integration of stress and reward information. There is an acute increase in NE in the BNST after restraint stress (44
) as well as an increase in DA during administration of drugs of abuse (42
). Therefore, our data indicate that both acute stress and drugs of abuse or reward may enhance excitatory transmission via activation of CRFR1 signaling. It is interesting to speculate that perhaps NE and DA are activating different populations of CRFR1, with DA enhancing CRF release from local dlBNST neurons and NE activating extrinsic CRF afferents to the regions (for example from the central nucleus of the amygdala).
Repeated cocaine disrupts CRFR1 signaling and decreases output from the dlBNST
In naive slices both DA receptor and β-AR signaling enhance excitatory transmission through CRFR1 actions in the dlBNST. There is evidence for downregulation of CRF signaling following repeated administration of drugs of abuse throughout the brain. For example, in the central nucleus of the amygdala (CeA) CRF release (53
) and mRNA levels (54
) are decreased after repeated administration of cocaine. Further, CRFR1 is internalized 30 minutes following a 14-day escalating dose morphine treatment (52
). Thus, the disruption of CRFR1 signaling that we observe after repeated cocaine administration likely reflects a downregulation of CRFR1 function. This raises the interesting possibility that NE signaling may be effectively re-routed through previously described α1
-ARs signaling, which leads to reductions in BNST excitatory transmission (27
Withdrawal from repeated cocaine sensitizes the CRF system in the dlBNST to cocaine challenge
While it appears that there is a downregulation of CRF signaling following repeated administration of drugs of abuse, there is evidence that an opposite phenomenon may occur during extended withdrawal. In rats in withdrawal from ethanol there is a marked increase in CRF release in the BNST as measured by microdialysis (56
). There is also an increase in CRF release in the CeA during opiate and cocaine withdrawal (53
). Moreover, Roberto and colleagues recently demonstrated that chronic ethanol exposure and withdrawal enhances CRF-dependent signaling in the CeA(58
). Our results show that while there may be increased CRF during withdrawal, CRF signaling remains intact as urocortin still increases sEPSC frequency. However, if a challenge of cocaine is given during this period CRFR1 signaling is once again disrupted.
A single injection of cocaine to drug-naïve animals produces no disruption of CRF signaling (see saline-cocaine animals, ) and in fact has been shown to enhance short term plasticity (25
), but animals that have previously received cocaine now have a disruption of CRF signaling with a single challenge injection. One possible explanation is that CRF signaling within the BNST may be highly sensitized during withdrawal. Perhaps in the presence of the higher levels of CRF that are present during withdrawal there is a change in the properties of the CRFRs that are returned to the membrane during the withdrawal state. CRFRs are known for promiscuous signaling (59
) and in fact in the lateral septal nucleus CRFR2 receptors shift from signaling through PKA to largely through PKC following chronic cocaine administration (60
). Another possibility is a shift in the relative number of CRFR1 and CRFR2 within the BNST following cocaine withdrawal. In the hippocampus, blocking CRFR1 but not CRFR2 attenuates LTP in naïve rats but these effects are reversed in rats in withdrawal from cocaine where now CRFR2 antagonism attenuates LTP (61
One or a combination of these mechanisms could produce BNST neurons that are highly sensitive to CRF-signaling. In this new state, when a cocaine injection is given which would presumably increase DA within the BNST (42
) CRF signaling may be rapidly activated, producing either a quick removal of CRF receptors from the membrane, a change in the type of CRF receptors present, or attenuation in their ability to be activated. Therefore, the ability to further activate this system is occluded and no effect is observed by the application of urocortin in animals 30 minutes after cocaine administration.
Implications for reinstatement models
The BNST, and specifically β-AR and CRF signaling within the BNST, are critically involved in stress-induced reinstatement models of drug seeking (18
). In the model proposed here, a stressor during the withdrawal period, which is known to increase NE release in the BNST, would be predicted to activate the system in a similar manner as a cocaine challenge. The disrupted state of CRF signaling by repeated administration and the subsequent highly-sensitized system that develops during withdrawal may be what is setting the molecular switch that is triggered for stressed-induced relapse. A drug challenge is likely activating other systems in addition to what is described by this model which are critical for drug priming induced reinstatement, such as CRF signaling within the accumbens (63
). This may in part explain why CRFR antagonism in the BNST has no effect on drug priming models of reinstatement (41
), yet an important effect on stress-induced reinstatement. Furthermore, this model projects a cellular mechanism for the finding that icv norepinephrine failed to induce reinstatement when it was preceded by a pretreatment of CRF antagonist (19
In conclusion, our results indicate a novel interaction between NE and CRF to enhance excitatory transmission within a region that plays a key role in mediating aspects of anxiety, reward, and relapse-related behaviors. Further, we describe how this system is disrupted and subsequently altered by repeated cocaine and withdrawal.