Dopamine produces activity-dependent enhancement of spontaneous glutamatergic transmission through D1- and D2-like dopamine receptors
We measured spontaneous excitatory postsynaptic currents (sEPSCs) recorded from neurons located in the dorsal-lateral BNST (dlBNST; ) (average basal frequency: 1.8 ± 0.4 Hz, average basal amplitude: 22 ± 2 pA, n = 16, representative trace shown in ). We focused on this particular region of the BNST based on previous findings that 1) neurons in this region have been shown to be activated by acute administration of drugs of abuse (Valjent et al., 2004
); 2) it receives a dopaminergic input () (Meloni et al., 2006
; Healey et al., 2008
); and 3) multiple substances of abuse promote robust rises in extracellular dopamine levels in this region (Carboni et al., 2000
). We focused our efforts on modulation of sEPSCs instead of evoked EPSCs because of the complex nature of the glutamatergic projections to the BNST. To test the hypothesis that dopamine modulates glutamatergic synaptic transmission in the dlBNST, dopamine (1μM) was bath applied for 5 minutes while spontaneous glutamatergic transmission was monitored using whole-cell patch clamp recordings in acutely prepared mouse brain slices. We found that this brief dopamine application resulted in a transient increase in the frequency of sEPSCs (198 ± 51% of basal frequency, p < 0.05, n = 10, ). Additionally, there was a modest, but significant effect on sEPSC amplitude (120 ± 10% of basal frequency, p < 0.05, n = 10, ) with no apparent effect on the kinetics of the sEPSC (normalized sEPSC trace shown inset). We then examined the ability of different concentrations of dopamine to modulate spontaneous glutamatergic transmission in the dlBNST. We found that dopamine exhibited a concentration-dependent enhancement of spontaneous glutamatergic synaptic transmission (; 100 nM dopamine, 100 ± 3% of basal frequency, n = 5; 300 nM, 130 ± 6% of basal frequency, n = 5; 50 μM, 165 ± 39% of basal frequency, n = 6, ; 100 nM dopamine, 98 ± 3% of basal amplitude, n = 5; 300 nM, 99 ± 2% of basal amplitude, n = 5; 50 μM, 109 ± 10% of basal amplitude, n = 6).
Dopamine enhances glutamatergic transmission in the dlBNST
Dopamine can act at adrenergic receptors (Malenka and Nicoll, 1986
) in addition to dopamine receptors. Thus, we wanted to establish that dopamine was enhancing spontaneous glutamatergic transmission in the dlBNST through activation of dopamine receptors. We found that pre-application of either a D1-like receptor antagonist (SCH23390, 3 μM, 94 ± 6% of basal frequency, n = 5) or a D2-like receptor antagonist (sulpiride, 1 μM, 101 ± 7% of basal frequency, n = 4, 10 μM, 113 ± 10% of basal frequency, n = 7, data not shown) prevented dopamine-induced increases in sEPSC frequency () and sEPSC amplitude (). Additionally, we found that neither SCH23390 (3 μM, 83 ± 14% of basal frequency, n = 4; 99 ± 7% of basal amplitude, n = 4) nor sulpiride (1 μM, 96 ± 5% of basal frequency, n = 4; 103 ± 5% of basal amplitude, n = 4) alone altered sEPSC amplitude or frequency, suggesting a lack of tonic activation of D1 and D2 like receptors in the dlBNST. Further, we found that in slices obtained from D1R KO mice, dopamine did not alter sEPSC frequency (99 ± 18% of basal frequency, n = 6, ) or sEPSC amplitude (106 ± 8% of basal frequency, n = 6, ). Taken together, the results obtained from these converging genetic and pharmacological approaches suggest that dopamine enhances spontaneous glutamatergic transmission in the dlBNST via activation of both D1- and D2-like receptors.
Activation of both D1-like and D2-like receptors is required for dopamine enhancement of spontaneous glutamatergic transmission in the dlBNST
In order to more clearly understand the mechanism of action of dopamine in the dlBNST, we next examined the ability of dopamine to modulate miniature EPSCs (mEPSCs). By examining the effect of a compound on mEPSCs, one can more precisely determine if the locus of action is pre- or postsynaptic. mEPSCs were isolated by addition of the sodium channel blocker tetrodotoxin (TTX, 1μM) to the bath solution (average basal frequency: 1.4 ± 0.4 Hz, average basal amplitude: 23 ± 1 pA, n = 10). Surprisingly, we found that in the presence of TTX, dopamine did not alter either mEPSC frequency (95 ± 5% of basal frequency, n = 5, ) or amplitude (95 ± 8% of basal frequency, n = 5 ). This suggests that dopamine is acting to alter glutamatergic transmission via an activity-dependent mechanism.
To further examine the activity-dependence of the dopamine regulation of excitatory transmission, we measured the ability of dopamine to modulate the excitability of neurons in the dlBNST using the current-clamp recording configuration (representative current-clamp recording shown in ). We found that dopamine had negligible effects on the resting membrane potential in the majority of the cells in the dlBNST (18/23) (). However, we found that in a subpopulation of neurons (5/23) dopamine application led to a marked transient depolarization (8.4 ± 0.5 mV, n = 5, p < 0.05) that partially reversed (2.4 ± 1.2 mV, n = 5)(), inducing spontaneous action potential firing in 3/5 neurons and increasing current-injection-induced firing of action potentials in 4/5 neurons. A comparison of the properties of the neurons that were robustly depolarized by dopamine compared to those that were not dramatically altered showed no significant differences in capacitance, input resistance, resting membrane potential or presence of hyperpolarization-activated current (IH
) (data not shown). The ability of dopamine to robustly depolarize a subpopulation of neurons in the dlBNST is similar to what has been previously noted with application of serotonin in current-clamp recordings in the dlBNST (Rainnie, 1999
Dopamine increases excitability in a subpopulation of neurons in the BNST.
Dopamine-induced enhancement of glutamatergic transmission requires CRF signaling
The combined findings that dopamine enhancement of glutamate transmission is activity dependent, and that only a subset of dlBNST neurons are robustly depolarized by dopamine suggest that dopamine could enhance glutamatergic transmission indirectly by depolarizing a subpopulation of dlBNST neurons to produce the release of a neurotransmitter or neuropeptide which then directly regulates glutamatergic transmission. Several studies, both functional (Meloni et al., 2006
) and anatomical (Phelix et al., 1994
), have demonstrated an interaction between dopamine and CRF in the BNST. Indeed, a subpopulation of neurons within the dlBNST are CRF positive (Day et al., 1999
; Rodaros et al., 2007
). Using dual-label fluorescent immunohistochemistry in colchicine-injected mice, we found that CRF-immunoreactive neurons in the dlBNST are closely apposed to tyrosine hydroxylase (TH) positive fibers (), consistent with previous findings obtained using electron microscopy (Phelix et al., 1994
CRF signaling is required for dopamine modulation of glutamatergic transmission in the BNST.
We hypothesized that dopamine alters glutamatergic transmission through activation of endogenous CRF signaling in the dlBNST. In keeping with this hypothesis we found that pre-application of the selective CRF-R1 antagonist NBI 27914 (Chen et al., 1996
)(1 μM) inhibited dopamine-induced increases in sEPSC frequency (96 ± 11% of basal frequency, n = 6, ). We next examined the ability of exogenously applied CRF to modulate glutamatergic transmission in the dlBNST. We found that bath application of 300 nM CRF increased sEPSC frequency (140 ± 14% of basal frequency, p < 0.05, n = 6, ) but not sEPSC amplitude (117 ± 15% of basal amplitude, n = 6, ). We then examined the ability of different concentrations of CRF to modulate spontaneous glutamatergic transmission in the dlBNST. We found that CRF exhibited a concentration-dependent enhancement of sEPSC frequency (; 100 nM CRF, 100 ± 3% of basal frequency, n = 5; 300 nM CRF, 130 ± 6% of basal frequency, n = 5; 1 μM CRF, 165 ± 39% of basal frequency, n = 6) but not sEPSC amplitude ().
Additionally, we examined the ability of Urocortin 1, an endogenous agonist of CRF receptors (Vaughan et al., 1995
), to modulate spontaneous glutamatergic transmission in the dlBNST. Similar to CRF, we found that bath application of 300 nM Urocortin 1 increased in sEPSC frequency (228 ± 40% of basal frequency, p < 0.05, n = 8, ) but not sEPSC amplitude (104 ± 5% of basal amplitude, n = 8, ).
Next we sought to determine the receptor subtype through which CRF is enhancing glutamatergic function in the dlBNST. We found that pre-application of NBI 27914 (1 μM) (97 ± 7% of basal frequency, n = 7, ), but not the CRF-R2 antagonist, Astressin-2B (100 nM) (107 ± 49% of basal frequency, p < 0.05, n = 5, ), blocked the ability of 300 nM CRF to enhance spontaneous glutamatergic transmission. Additionally, we found that CRF did not alter sEPSC amplitude in the presence of either the CRF-R1 or the CRF-R2 antagonists, (). Further, we found that the ability of CRF to enhance spontaneous glutamatergic transmission persisted in the presence of SCH23390 (10 μM, 150 ± 14 % of basal frequency, p < 0.05, n = 4).
CRF acts through CRF-R1 receptors to enhance glutamate release in the dlBNST
There have been previous reports that CRF receptors can be tonically active in slice preparations (Liu et al., 2004
). We examined the tonic-activity of CRF receptors in the dlBNST in our slice preparation by examining the ability of either NBI27914 or Astressin2B alone to modulate spontaneous glutamatergic transmission. Interestingly, we found that application of NBI 277914 led to a modest but significant reduction of sEPSC frequency (90 ± 3% of basal frequency, n = 4) but not amplitude (102 ± 4% of basal amplitude, n = 4) whereas Astressin-2B had no effect on spontaneous glutamatergic transmission (101 ± 10% of basal frequency, n = 5; 104 ± 5% of basal amplitude, n = 5). This suggests that CRF-R1 may tonically regulate glutamate release in our dlBNST slice preparation.
We then sought to determine the mechanism by which CRF was enhancing glutamatergic function in the dlBNST by examining CRF modulation of mEPSCs. In contrast to dopamine, we found that in the presence of TTX, CRF enhanced mEPSC frequency (145 ± 8 % of basal frequency, p < 0.05, n = 5, inset ) but not amplitude (100 ± 2 % of basal amplitude, n = 5, ). Taken together, these results suggest that CRF is enhancing glutamate release through activation of CRF-R1 in the dlBNST.
In vivo recruitment of catecholaminergic signaling in the BNST induces a NMDA-receptor dependent enhancement of Short-Term Potentiation (STP)
Both dopamine (Gao et al., 2006
; Navakkode et al., 2007
) and CRF (Blank et al., 2002
; Ungless et al., 2003
) have been previously suggested to modulate NMDAR-dependent synaptic plasticity in other brain regions. In particular, in the hippocampus, CRF has been suggested to “prime” synapses for LTP induction (Blank et al., 2002
). In order to examine the effect of dopamine and CRF on synaptic plasticity in the dlBNST, we performed field recordings, as we have previously shown a robust form of stimulus induced LTP (Weitlauf et al., 2004
). To begin to determine the consequences of in vivo
elevation of extracellular dopamine levels in the BNST on glutamate synapses, we gave mice a single intraperitoneal (ip) injection of cocaine (20 mg/kg) or saline after four days of habituating saline injections in a blinded design. 30 minutes after injection of cocaine or control saline, brain slices were prepared and electrophysiological recordings were performed as previously described to examine effects on stimulus-evoked plasticity in the BNST (Weitlauf et al., 2004
Local stimulation of the BNST elicits an extracellular field response consisting of two prominent downward deflections, much like in the striatum, which we refer to as N1 and N2 (, inset). The N2, but not the N1, is eliminated by CNQX, suggesting that this portion is AMPAR mediated (Weitlauf et al., 2004
; Grueter and Winder, 2005
). Stimulation of glutamatergic afferents to the BNST with a moderate stimulus protocol (two 100 Hz, 1 second trains with a 20 second interstimulus interval) elicits significant, stable, enhancement of the N2 without effect on the N1 (, circles, n=12). This enhancement is NMDAR-dependent (, closed squares, n=8) as previously described (Weitlauf et al., 2004
). In animals receiving cocaine there was transient (approximately 20 minutes) enhancement of the field potential post-tetanus, which we refer to as short-term potentiation (STP) (Area under curve from minutes 0–20 post-tetanus: saline, 517 ± 104 relative units, n = 12 from 8 saline treated animals; cocaine, 1097 ± 193 relative units, n = 7 from 6 cocaine treated animals, p < 0.01, ).
Cocaine produces an enhancement of NMDAR-dependent plasticity following tetanization in the dlBNST.
Dopaminergic signaling within the dlBNST mediates cocaine-enhancement of STP
The in vivo
effects of cocaine on subsequent ex vivo
STP in BNST slices could reflect local actions of cocaine on catecholamine fibers in the BNST, or it could result from larger network effects of the in vivo
cocaine. To differentiate between these possibilities, we prepared slices containing the BNST from naïve mice, and bath applied cocaine prior to high-frequency stimulation. Addition of 3 μm cocaine for 30 minutes did not alter basal transmission ( squares n=9); however, this cocaine application produced an enhancement of STP when a tetanus was given 60 minutes following washout (, n=9; F=14.37, p<0.01). This enhancement was specific to STP as LTP measured 50 minutes post-tetanus was not altered by cocaine (Supplementary Figure 1C
, p=0.45). Blockade of NMDA receptors with DL-APV (100 μM)
similarly attenuated LTP whether cocaine was pre-applied or not (, n=8 each; ), suggesting that cocaine-induced enhancement of STP is NMDAR-dependent. Thus, the effect of ex vivo
cocaine on STP was similar to the effects of in vivo
In addition to dopaminergic input to the BNST there are also other monoaminergic inputs, including a dense projection of noradrenergic fibers from nucleus tractus solitarius (NTS) through the ventral noradrenergic bundle (VNAB) (Delfs et al., 2000
) as well as a serotonergic input from the midbrain raphe nuclei (Commons et al., 2003
). Because cocaine can act at multiple transporters, it is possible that the effect on STP could be mediated independently of dopamine. In order to further examine the role of dopamine in mediating enhanced plasticity in the dlBNST, we examined the ability of the DAT-selective antagonist, GBR12909, to alter STP. Consistent with our previous results, we found that bath application of GBR12909 prior to induction of LTP (time course of application shown in ) lead to an enhancement of STP (p < 0.05, n = 8 control, n= 6 GBR12909, ). To further determine whether the dopamine system was responsible for cocaine-induced enhancement of STP we applied the pan-dopamine receptor antagonist flupenthixol (10 μM) with cocaine and observed that enhancement of STP was blocked ( flupenthixol and cocaine n=7, cocaine n=16; F=6.43, p<0.05). We next examined the role of dopaminergic signaling in the mediation of the effects of in vivo
cocaine administration by examining the ability of cocaine to alter STP in D1R KO mice. Consistent with our results obtained performing ex vivo
cocaine application, we found that STP was not significantly enhanced in D1R KO mice following injection of cocaine compared to saline injected mice (, n=7 from 4 cocaine treated animals, n=4 from 3 saline treated animals).
Cocaine-induced enhancement of plasticity is dependent on dopamine and CRFR1 signaling
Based on our observation that CRF-R1 activation was necessary to produce dopaminergic enhancement of spontaneous glutamatergic transmission, we tested whether the cocaine enhancement of STP may also act via CRF-R1 signaling. Thus we co-applied NBI-27914 (1μM) in conjunction with cocaine and found that this manipulation also blocked cocaine-induced enhancement of STP ( NBI-27914 and cocaine n=7, cocaine n=16; F=6.43, p<0.05).