Two Functional States of NAc Neurons in One-day-old Slice Cultures
In one-day-old slice cultures, NAc neurons maintained their characteristic spiny morphology () and electrophysiological properties (1
). Whole-cell current-clamp recordings revealed that ~45% of NAc neurons (n
= 48/104) periodically oscillated between two distinct membrane potentials commonly referred to as the upstate and downstate (4
). Analysis of the distribution of membrane potentials during upstates and downstates indicated that the average downstate potential was −75 mV (±6 mV, n
= 24), while the average upstate potential was −62 mV (±5 mV, ). These membrane potentials are similar to those seen during upstates and downstates in vivo
CREB influences the upstate of NAc neurons in slice cultures
Both in vitro
and in vivo
observations suggest that the upstate potential is generated by bursting/synchronous glutamatergic synaptic inputs (4
). We therefore first examined whether the upstate potential is mediated by glutamatergic synaptic inputs in the slice culture preparation. The two types of glutamate receptors that mediate most fast excitatory synaptic transmission are α
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type receptors (AMPARs) and NMDARs. Under physiological conditions, activation of NMDARs requires the prior activation of AMPARs to depolarize the membrane potential. Thus, blocking AMPARs can functionally block activation of both AMPARs and NMDARs. In slice cultures, the upstate of NAc neurons was abolished by application of the AMPAR antagonist NBQX (3 μ
M, , n
= 5), indicating that the upstate of NAc neurons is mediated by activation of AMPARs and/or NMDARs. We next examined whether the excitatory synaptic activity in the upstate-downstate cycling NAc neurons has a bursting pattern, because only bursting synaptic activity can be temporally summated to generate a stable depolarization plateau of the membrane potential (i.e.
the upstate membrane potential plateau). To test this, we used whole-cell voltage-clamp techniques to record spontaneous EPSCs (sEPSCs) in NAc neurons. We found that sEPSCs for the upstate neurons indeed occurred in a bursting pattern (, n
= 5). These observations suggest that the upstate of NAc neurons in slice culture is mediated by bursting sEPSCs.
Activation of CREB Prolongs the Upstate of NAc Neurons
Exposure to drugs of abuse has been shown to alter the bimodal states (i.e.
up- and downstate) of NAc neurons in vivo
). A common molecular change in NAc neurons following exposure to drugs of abuse is the activation of the transcription factor CREB (2
). To examine whether activation of CREB influences the bimodal property of NAc neurons, we manipulated CREB activity within individual neurons using virus-mediated gene transfer to express caCREB or dnCREB tagged with GFP. As reported previously (1
), both caCREB and dnCREB readily entered the nucleus of infected neurons (). Expression of either CREB constructs or GFP alone did not affect the percentage of neurons exhibiting bimodal states (43–50% of neurons, n
> 40 for each manipulation). However, the duration of individual upstates was significantly longer in NAc neurons expressing caCREB-GFP than in either dnCREB-expressing or uninfected neurons (, in seconds: uninfected, 1.0 ± 0.3, n
= 21; caCREB, 1.9 ± 0.4, p
< 0.05, n
= 17; dnCREB, 1.0 ± 0.4, n
= 12). In addition, the number of action potentials observed during individual upstates was significantly increased by expression of caCREB-GFP and decreased by expression of dnCREB-GFP (, uninfected, 1.5 ± 0.3, n
= 21; caCREB, 2.8 ± 0.7, p
< 0.05, n
= 17; dnCREB, 0.6 ± 0.5, p
< 0.05, n
= 12). In contrast, neither the frequency (number of upstates per min: uninfected, 17.3 ± 6.2, n
= 21; caCREB, 22.5 ± 8.6, n
= 17; dnCREB, 15.2 ± 12.7, n
= 12, ) nor the amplitude (in mV: uninfected, 16.8 ± 1.9, n
= 21; caCREB, 18.5 ± 4.1, n
= 17; dnCREB, 17.1 ± 4.8, n
= 12, ) of individual upstates was significantly altered by expression of caCREB or dnCREB.
Activation of CREB Selectively Enhances NMDAR-mediated Synaptic Current
Because AMPAR- and NMDAR-mediated synaptic currents are the driving force for the upstates of NAc neurons (4
), we next examined whether the CREB effect on upstates results from changes in AMPAR or NMDAR EPSCs. We performed pairwise comparisons of both AMPAR- and NMDAR-mediated EPSCs between infected and adjacent uninfected NAc neurons using identical afferent stimulation (12
). Briefly, we first made a whole-cell voltage-clamp recording from one cell (either infected or uninfected) and held the membrane potential at −60 mV. Because of the voltage-dependent Mg2+
blockade of NMDARs, these receptors were minimally activated at this holding potential. The recorded EPSCs were therefore primarily mediated by AMPARs (AMPAR EPSCs). Then, without changing the parameters for presynaptic stimulation, the membrane potential was switched to +40 mV. Because this depolarized potential removes the voltage-dependent Mg2+
blockade, NMDARs could be activated and together with AMPARs mediated dual-component EPSCs (AMPAR EPSC + NMDAR EPSC). Because of the fast inactivation of AMPAR EPSCs, the dual-component EPSC at 30 ms after onset was mainly mediated by NMDARs (), and thus was operationally measured as the amplitude of NMDAR EPSCs. Therefore, both AMPAR and NMDAR EPSCs could be measured from the first cell by changing the holding potentials. Immediately after finishing recording from the first neuron, without changing the presynaptic stimulation, we made the same measurements from an adjacent cell of opposite phenotype (i.e.
uninfected or infected; the sequence in which recordings were made was alternated during the course of experiments). By keeping the stimulation parameters constant, roughly the same set of presynaptic fibers was stimulated to activate similar numbers of synapses on adjacent pairs of neurons. When normalized to the peak amplitude of EPSCs in adjacent uninfected neurons, the AMPAR EPSCs were not significantly altered by expression of either GFP (1.01 ± 0.18, n
= 6), caCREB (1.05 ± 0.15, n
= 14), or dnCREB (0.99 ± 0.14, n
= 11) ().
The paired-pulse ratio (PPR, 2nd:1st) is often used as a sensitive measurement to detect changes in presynaptic release. The PPR (interpulse interval = 200 ms) of AMPAR EPSCs was not affected by expression of caCREB (uninfected, 0.93 ± 0.07; caCREB, 0.98 ± 0.04, n = 14) or of dnCREB (uninfected, 0.88 ± 0.04; dnCREB, 1.01 ± 1.22, n = 11, ), suggesting no significant presynaptic alterations.
Another sensitive electrophysiological assay to detect changes in presynaptic release and postsynaptic responsiveness is the measurement of miniature (m) EPSCs. In general, a change in the frequency of mEPSCs reflects a change in presynaptic release, whereas a change in the amplitude reflects a change in postsynaptic responsiveness. In the presence of tetrodotoxin (TTX), which blocks spontaneous action potentials in presynaptic terminals, we measured mEPSCs in uninfected and infected NAc neurons and observed that neither the amplitude (in pA: uninfected, 16.5 ± 2.2, n = 22; caCREB, 17.2 ± 1.9, n = 14; dnCREB, 16.3 ± 3.1, n = 15), nor the frequency (in Hz: uninfected, 24.9 ± 4.1, n = 22; caCREB, 24.1 ± 5.2, n = 14; dnCREB, 22.3 ± 3.8, n = 15), of AMPAR-mediated mEPSCs was significantly altered by manipulations of CREB activity (). These findings suggest that neither the presynaptic release of glutamate nor postsynaptic AMPAR function were altered by expression of caCREB or dnCREB.
In contrast to AMPAR EPSCs, NMDAR EPSCs were substantially altered by changes in CREB activity. NAc neurons expressing caCREB had significantly larger NMDAR EPSCs than control neurons, whereas expression of either GFP alone or dnCREB had no significant effects (peak amplitude relative to adjacent uninfected neurons: GFP, 0.89 ± 0.15, n = 6; caCREB, 1.57 ± 0.19, n = 14, p < 0.01; dnCREB, 0.88 ± 0.11, n = 15, ). Because CREB activation selectively up-regulates NMDAR EPSCs without affecting AMPAR EPSCs, the ratio of AMPAR:NMDAR should be lower in caCREB-expressing neurons than in uninfected control neurons. This prediction was also confirmed in the pairwise recordings (uninfected, 0.82 ± 0.12; caCREB, 0.54 ± 0.16; n = 14, p < 0.05, paired Student’s t test).
Two of the most likely mechanisms through which caCREB could increase NMDAR EPSCs are an increase in the function of pre-existing synaptic NMDARs or an addition of new NMDARs on the cell surface. To address the latter possibility, we examined whether expression of caCREB-GFP in NAc neurons affected the surface or total level of the NMDAR subunit NR1. Because functional NMDARs require NR1, surface expression of NR1 reflects the total number of functional NMDARs on the plasma membrane (21
). In this set of experiments, we first dissected the NAc tissue and briefly (10 min) incubated it with viral vectors before placing it on the tissue culture film. Twenty-four hours later, >90% of the NAc neurons were infected (indicated by their GFP signals, data not shown). We then performed Western blot analysis of biotinylated surface NR1 subunits in NAc neurons (see “Experimental Procedures”), which revealed a significant increase induced by expression of caCREB but not dnCREB nor GFP alone (relative intensity of NR1 protein bands: GFP, 1.00 ± 0.15, n
= 7; dnCREB, 1.05 ± 0.163, n
= 8; caCREB, 1.89 ± 0.21, n
= 8, p
< 0.01, ). The total level of NR1 subunits was not significantly altered by any of these manipulations (relative density: GFP, 1.00 ± 0.04, n
= 7; dnCREB, 0.95 ± 0.08, n
= 8; caCREB, 1.16 ± 0.09, n
= 10). Accordingly, the ratio of surface NR1 to total NR1 was increased by caCREB expression (normalized to the ratio of GFP group: dnCREB, 1.10 ± 0.20, n
= 7; caCREB, 2.09 ± 0.30, n
= 8, p
< 0.01, ). The increase in surface but not total NR1 suggests that caCREB likely influences NMDAR trafficking to the surface or their stability on the plasma membrane (21
NMDARs Mediate CREB-induced Enhancement of Upstate of NAc Neurons
Although the AMPAR- and NMDAR-mediated synaptic currents together mediate the upstate of NAc neurons, their contributions are different. Computational studies suggest that AMPAR-mediated synaptic inputs preferentially mediate the initiation of the upstate, whereas NMDAR-mediated synaptic inputs preferentially mediate the elongation of the upstate (24
). Given our observations that caCREB selectively prolongs the duration of upstates () and that caCREB up-regulates surface NMDARs ( and ), we hypothesized that the enhancement of NMDAR EPSCs by caCREB is sufficient to lead to prolonged upstate duration. To test this hypothesis, we first examined whether NMDARs indeed contribute to the duration of upstates of NAc neurons in the slice culture preparation. As shown in , in uninfected NAc neurons, acute application of the NMDAR antagonist D-APV (50 μ
M) significantly shortened the duration of upstates (relative change, 29.4 ± 8.1%, n
= 7, p
< 0.05), indicating that NMDARs influence the duration of upstates of NAc neurons in slice cultures.
NMDARs mediate the CREB effect on upstate duration, but not the upstate action potential firing
We next examined whether up-regulation of NMDARs is the primary mechanism by which caCREB prolongs the upstates of NAc neurons. In caCREB-expressing neurons, acute inhibition of NMDARs by D-APV abolished the increase in the duration of the upstate (in seconds: uninfected, 0.87 ± 0.13, n = 12; caCREB, 0.84 ± 0.29, n = 11; dnCREB, 0.85 ± 0.18, n = 11, ). This result suggests that the effect of increased CREB activity on the upstate duration is mediated by NMDARs.
In contrast to the significant effect on upstate duration, application of D-APV did not affect either the caCREB-induced up-regulation or the dnCREB-induced suppression of action potential firing during the upstate (number of action potentials per upstate in APV: uninfected, 1.26 ± 0.42, n
= 2; caCREB, 2.28 ± 0.58, n
= 11, p
< 0.05; dnCREB, 0.52 ± 0.41, n
= 11, p
< 0.05, ). This result suggests that the effect of CREB on action potential firing is independent of activation of NMDARs. Indeed, previous work has shown that caCREB expression increases the excitability of NAc neurons by regulating a set of voltage-gated ion channels (1
). To further test the role or lack thereof of NMDARs in CREB-mediated regulation of action potential firing, we examined evoked action potential firing by depolarizing current pulses in the presence of D-APV. As shown in , the caCREB-induced enhancement of action potential firing in NAc neurons was not affected by application of D-APV (number of action potentials elicited by 100 pA: uninfected, 4.9 ± 0.9, uninfected-APV, 5.1 ± 0.7, n
= 12; caCREB, 9.8 ± 1.1, caCREB-APV, 10.0 ± 1.6, n
= 11; by 200 pA: uninfected, 18.2 ± 2.1, uninfected-APV, 17.2 ± 2.4, n
= 12; caCREB, 20.8 ± 1.4, n
= 11, caCREB-APV, 21. 4 ± 1.1, n
= 10, p
< 0.05, ).
Taken together, these observations suggest that CREB activation increases both the upstate duration and action potential firing in NAc neurons but does so via distinct molecular mechanisms within NAc neurons: activation of CREB increases upstate duration by enhancing NMDAR-mediated synaptic transmission and increases membrane excitability by modulating voltage-gated ion channels (1
Mimicking CREB-induced Up-regulation of NAc NMDARs Suppresses Locomotor Activity in Rats
We next addressed whether the up-regulation of NMDAR-mediated synaptic transmission in the NAc contributes to the behavioral consequences of increased CREB activity in this brain structure. One widely used approach to stimulate NMDAR-mediated functions is to inject NMDA into the NAc. However, injection of NMDA activates NMDARs in the absence of endogenously released glutamate, often resulting in nonspecific effects. Instead, we used DCS, which is a co-agonist of NMDARs; it binds to the glycine site of NR1 subunits and potentiates the function of NMDARs only when they are also bound to endogenous glutamate (25
). This approach minimizes the potential nonspecific effects that can accompany application of NMDA itself.
To validate this approach, we examined the effect of DCS on NMDAR EPSCs of NAc neurons in acute slices. Whole-cell voltage-clamp recordings were made at −30 mV in the presence of AMPAR and GABAA
R antagonists. After obtaining stable NMDAR EPSCs, we switched to DCS (10 μ
M)-containing bath solution and observed a significant increase in the peak amplitude of NMDAR EPSCs (). Furthermore, the potentiated currents were blocked by the NMDAR-selective antagonist D-APV (50 –100 μ
M) (), indicating that the DCS-enhanced currents were mediated by NMDARs. Having verified the effectiveness of DCS, we injected it, using a behaviorally relevant dose (10 μ
), into the NAc shell of rats through chronic guide canulae () and examined locomotor activity under three different conditions: spontaneous locomotor activity, novelty-induced locomotor activity, and cocaine-induced locomotor activity. Spontaneous locomotor activity reflects the basal motor function of animals, whereas novelty- and drug-induced locomotor enhancement have been related to the sensitivity of animals to the reinforcing effects of addictive drugs (27
). Bilateral intra-NAc injections of DCS alone did not affect spontaneous locomotor activity (). However, rats that received these same injections prior to placement in the activity monitors exhibited significantly reduced novelty-induced locomotor activity compared with animals that received intra-NAc injections of saline (p
< 0.05, F1, 22 = 5.741, ANOVA, n
= 13 and 11 for saline and DCS group, respectively; ). Furthermore, cocaine-induced locomotor activity was also significantly lower following intra-NAc injections of DCS compared with saline-injected controls (p
< 0.05, F1, 22 = 6.467, ANOVA, n
= 10 and 14 for saline and DCS group, respectively; ). These results suggest that the effect of CREB activation on NMDARs, and thus the upstates of NAc neurons, may act to decrease the sensitivity of animals to drug-related stimulation.
Up-regulation of NAc NMDARs suppresses novelty- and cocaine-induced locomotor activity
Activation of CREB in the NAc is a common molecular adaptation following exposure to drugs of abuse. An evolving body of evidence shows that activation of CREB in NAc suppresses addictive behaviors (1
), but the underlying mechanism remains largely unclear. The CREB-NMDAR-upstate pathway identified in this study may provide a mechanism that links the cellular effect of CREB to its overall inhibitory role in regulating drug responsiveness.