Corticotropin-releasing factor in the nucleus accumbens shell induces swim depression, anxiety, and anhedonia along with changes in local dopamine/acetylcholine balance.

The nucleus accumbens shell (NAcS) has been implicated in controlling stress responses through corticotropin-releasing factor (CRF). In addition to studies indicating that CRF in the NAcS increases appetitive motivation, there is indirect evidence suggesting that NAcS CRF may also cause aversive responses and that these behaviors may be mediated through local dopamine (DA) and acetylcholine (ACh) systems. To provide a direct test of this hypothesis, we used male Sprague-Dawley rats with implanted cannulas aimed at the NAcS. Experiment 1 showed local CRF injection (10 or 50 ng/side) to increase immobility in the forced swim test and a CRF antagonist D-Phe-CRF ((12-41)) to attenuate this depressive-like behavior. In Experiment 2, injection of CRF (250 ng/side) also decreased the rats' preference for sucrose, while in Experiment 3, CRF (50 or 250 ng/side) induced anxiety-like behaviors in an elevated plus maze and open field. These same doses of CRF in Experiment 4 failed to alter the rats' locomotor activity, indicating that these behavioral changes were not caused by deficits in activity. In Experiment 5, results from in vivo microdialysis revealed that CRF in the NAcS markedly increased local extracellular ACh, while also producing a small increase in DA. These results show that NAcS CRF can generate a variety of aversive behaviors, including swim depression, anhedonia, and anxiety, in addition to approach behavior. They suggest that these behaviors may occur, in part, through enhanced activation of ACh and DA in the NAcS, respectively, supporting a role for this brain area in mediating the dual effects of stress.

Aversive hypothalamic stimulation releases acetylcholine in the nucleus accumbens, and stimulation-escape decreases it.

Hypothalamic electrodes can generate positive reinforcement, as shown by self-stimulation, and negative reinforcement shown by stimulation-escape. It was hypothesized that acetylcholine (ACh) is released in the nucleus accumbens during the aversive state that underlies stimulation-escape. If this is correct, escape behavior should lower extracellular ACh. Rats were prepared with microdialysis probes in the accumbens (posterior shell region) and electrodes in the perifornical lateral hypothalamus. Animals learned to press a lever for 0.5 s trains of stimulation (typically 3600 responses/h). Then they were given automatic stimulation to determine which animals would also learn to press a lever to turn stimulation off for 5 s at a time (typically 75 responses/h). Accumbens microdialysis showed that automatic stimulation caused extracellular ACh to double, but only in the rats that were motivated to learn stimulation-escape. When allowed to escape stimulation, these animals lowered extracellular ACh significantly. It is concluded that ACh release in the accumbens is related to the neural state that animals work to escape.

Supraadditive effect of d-fenfluramine plus phentermine on extracellular acetylcholine in the nucleus accumbens: possible mechanism for inhibition of excessive feeding and drug abuse.

The combination of d-fenfluramine plus phentermine (d-FEN/PHEN) provides a tool for exploring neural mechanisms that control food intake and drug abuse. Prior research suggests that dopamine (DA) in the nucleus accumbens can reinforce appetitive behavior and acetylcholine (ACh) inhibits it. When rats were given d-fenfluramine (5 mg/kg, IP) DA increased to 169% (p < 0.01), and ACh decreased slightly. Phentermine (5 mg/kg, IP) increased extracellular DA to 469% of baseline and ACh increased slightly to 124% (both p < 0.01). The d-FEN/PHEN combination, however, increased both DA and ACh with a supraadditive effect on ACh to 172%. One interpretation is that dFEN/PHEN increases DA like a meal or drug of abuse, while also increasing ACh to stop further approach behavior. This leaves the animal "satiated," as defined by reduced intake of food or drugs.

Acetylcholine release in ventral tegmental area by hypothalamic self-stimulation, eating, and drinking.

Evidence is presented for an acetylcholine (ACh) input to the midbrain ventral tegmental area (VTA) as part of a system for self-stimulation and ingestive behavior. Male rats were prepared with an electrode in the perifornical lateral hypothalamus and an ipsilateral guideshaft for microdialysis in the VTA. Extracellular ACh increased in the VTA during self-stimulation, auto-stimulation, eating, or drinking. Infusion of atropine into the VTA via the microdialysis probe was sufficient to stop self-stimulation and reduce intake of food. It is concluded that ACh acts at muscarinic receptors in the VTA as part of a circuit that modulates hypothalamic self-stimulation and ingestive behavior.

Dopamine release in the nucleus accumbens by hypothalamic stimulation-escape behavior.

It is known that lateral hypothalamic stimulation or self-stimulation can release dopamine in the nucleus accumbens (NAc). The present experiment illustrates that an aversively motivated behavior can also do this. Rats were prepared with microdialysis probes in the NAc and electrodes in the lateral hypothalamus (LH) or medial hypothalamus (MH). Automatic stimulation of the LH increased extracellular dopamine in the NAc 30% as reported earlier. The animals would perform both self-stimulation to turn the current on and stimulation-escape to turn it off, suggesting a combination of reward and aversion. Escape responding increased extracellular dopamine (DA) 100%, even though there was less total stimulation. Automatic stimulation of the MH did the opposite of the LH by decreasing accumbens dopamine (-20%), and the animals would only perform stimulation-escape, indicative of pure aversion. But again, extracellular DA in the NAc increased 100% during escape responding. Thus DA can be released during negative reinforcement when an animal's behavior is reinforced by escape from lateral or medial hypothalamic stimulation. This suggests that DA release was correlated with stimulation-escape behavior, rather than the aversiveness of automatic stimulation.

Norepinephrine microinjections in the hypothalamic paraventricular nucleus increase extracellular dopamine and decrease acetylcholine in the nucleus accumbens: relevance to feeding reinforcement.

Norepinephrine (NE) was microinjected into the paraventricular nucleus (PVN), while microdialysis was used to monitor extracellular dopamine (DA) and acetylcholine (ACh) in the nucleus accumbens (NAc). The PVN is a site where exogenously administered NE can act through alpha 2 receptors to elicit eating behavior and preference for carbohydrates. It was hypothesized that NE in the PVN acts on a behavior reinforcement system by altering the DA/ACh balance in the NAc. NE microinjections (80 nmol in 0.3 microliter), which effectively elicited feeding in satiated rats in a separate test, caused a significant increase in extracellular DA (109%) and decrease in ACh (-27%) when the same animals were tested in the absence of food. In contrast when the food was available and ingested, ACh increased (51%) instead of decreasing. These results support the hypothesis that a functional link exists between the PVN and the NAc in which DA helps initiate and ACh helps stop appetitive behavior involved in the reinforcement of eating.

Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala.

A number of experimental results has pointed to a cholinergic involvement in the stress response. Recently, analytical techniques have become available to measure acetylcholine release in vivo during exposure to various stressors. In these experiments, microdialysis was used to monitor acetylcholine output every 15 min in the dorsal hippocampus, amygdala, nucleus accumbens and prefrontal cortex before, during and after 1 h of restraint, including a 15-min session of intermittent tail-shock (1/min, 1 mA, 1-s duration) in rats. In response to the stressful event, acetylcholine release was significantly increased in the prefrontal cortex (186%; p < 0.01) and hippocampus (168%; P < 0.01) but not in the amygdala or nucleus accumbens. The sole effects observed in the amygdala and nucleus accumbens occurred upon release from the restrainer, at which point acetylcholine levels were significantly elevated in both areas (amygdala: 150%; P < 0.05; nucleus accumbens: 13%; P < 0.05). An enhanced acetylcholine release was also evident during this sample period in the hippocampus and prefrontal cortex. These data demonstrate an enhancement of cholinergic activity in response to stress in two acetylcholine projection systems (hippocampus and prefrontal cortex) but not in the intrinsic acetylcholine system of the nucleus accumbens or the extrinsic innervation of the amygdala. Moreover, the data showed that relief from stress was accompanied by a more ubiquitous acetylcholine response that extended to each site tested.

Morphine and naloxone, i.p. or locally, affect extracellular acetylcholine in the accumbens and prefrontal cortex.

In rats with microdialysis probes in the nucleus accumbens (NAc) or prefrontal cortex (PFC), intraperitoneally (IP) delivered morphine on the 8th day of escalating doses decreased extracellular ACh in the NAc. On day 9, naloxone (5 mg/kg) precipitated withdrawal and increased the release of ACh. When morphine and methylnaloxonium were given locally into the NAc by reverse dialysis, the opiate again decreased extracellular ACh, and the opiate antagonist increased it. These effects were proportional to the dose of local infusions. Local morphine had the same ACh-lowering effect in morphine-dependent and nondependent rats, whereas local methylnaloxonium increased extracellular ACh significantly more in morphine-dependent animals. Systemic and local effects on ACh systems in the PFC were more complicated and showed some relation to locomotor activity. The results suggest that intrinsic ACh neurons in the NAc have a special relationship to opiate reinforcement such that extracellular ACh is low in response to morphine and high during withdrawal. Thus, low ACh may correlate with opiate reward, and high ACh with aversion.

Extracellular acetylcholine is increased in the nucleus accumbens following the presentation of an aversively conditioned taste stimulus.

To determine if acetylcholine (ACh) is released in the nucleus accumbens in response to a conditioned stimulus (CS) that reminds the animal of an aversive event, in vivo microdialysis was used to monitor extracellular ACh during conditioned taste aversion. Saccharin flavored water (2.5 mM saccharin) was paired twice with nausea induced by i.p. lithium chloride (100 mg/kg). This is normally sufficient to create an aversion to the taste of saccharin, but instead of a preference test, the saccharin solution was squirted directly into the rat's mouth via a cheek catheter during nucleus accumbens microdialysis. The result was a 40% increase in extracellular ACh. We reported earlier that dopamine changes in the opposite direction; it decreases. This suggests that high synaptic ACh and low DA are correlated with an aversive state and cessation of behavior.

An appetitively conditioned taste elicits a preferential increase in mesolimbic dopamine release.

Rats were prepared with intragastric (IG) cannulae for infusing a nutrient into the stomach and microdialysis guide shafts in the nucleus accumbens (NAC) and striatum (STR) for measuring changes in extracellular dopamine. Prior to dialysis, subjects were trained to prefer the mildly bitter taste of sucrose octaacetate (SOA; CS+) by pairing voluntary intake with automatic IG infusions of nutritive polycose. The mildly sour taste of citric acid (CS-) was paired with IG water infusions as a control. Unconditioned animals received four exposures to SOA and citric acid on counterbalanced, alternating days. After training, dialysis samples were collected every 30 min before, during, and after intake of the CS+ or CS- in response to 14 h water deprivation on counterbalanced, consecutive days. Voluntary intake of the CS+ for 30 min significantly increased extracellular DA in the NAC but not in the STR of conditioned subjects. Intake of the CS- did not alter DA efflux at either site. Unconditioned, control rats also showed no DA response to either taste. These results show selective activation of the mesolimbic dopaminergic projection system as a consequence of a conditioned taste stimulus paired with a nutritive gastric load. This suggests that conditioned DA release may play a role in learned ingestive behavior based on the postingestive effects of food.

Effects of supplemental choline on extracellular acetylcholine in the nucleus accumbens during normal behavior and pharmacological acetylcholine depletion.

Brain microdialysis was used to determine whether systemic or local application of choline would modify the extracellular concentration of acetylcholine (ACh) in the nucleus accumbens (NAc) of freely moving rats. Supplemental choline given intraperitoneally or into the NAc of normal rats did not increase extracellular ACh. When local ACh interneurons in the NAc were treated pharmacologically to deplete the intracellular stores of ACh, then systemic choline (80 mg/kg) was an effective treatment. Specifically, 1) blockade of the high-affinity choline transporter with hemicholinium-3 (HC-3) to reduce ACh synthesis caused a decrease in extracellular ACh, but choline supplementation restored ACh toward its normal level in the NAc. 2) Local bicuculline treatment released ACh to the point of depletion, but systemic choline or locally infused choline helped maintain normal ACh levels. These results suggest that choline supplementation might be useful in preventing depletion of ACh in the nucleus accumbens during pathological conditions.

In vivo modulation of acetylcholine in the nucleus accumbens of freely moving rats: II. Inhibition by gamma-aminobutyric acid.

Our earlier studies suggest dopamine and serotonin interact with acetylcholine (ACh) in the nucleus accumbens (NAC) as part of a system for motivation and reinforcement. The purpose of the present experiment was to characterize a possible link between GABA and acetylcholine in the nucleus accumbens using microdialysis in freely moving rats. Different doses of GABA, muscimol, baclofen, saclofen and bicuculline were locally infused into the nucleus accumbens through the microdialysis probe. GABA and its agonists dose-dependently decreased extracellular levels of acetylcholine in the nucleus accumbens. In contrast the GABAA antagonist, bicuculline, dose-dependently increased extracellular ACh while the GABAB antagonist, saclofen, was without effect. Co-infusion of bicuculline or saclofen was shown to block the decrease in recoverable ACh produced by muscimol or baclofen, respectively. The results demonstrate an inhibitory action of GABA on acetylcholine interneurones in the nucleus accumbens involving both GABAA and GABAB receptor subtypes. In addition a tonic inhibitory GABAergic tone is probably mediated through GABAA receptors.

In vivo modulation of acetylcholine in the nucleus accumbens of freely moving rats: I. Inhibition by serotonin.

Microdialysis was used to characterize the effect of serotonergic input on cholinergic interneurons in the nucleus accumbens (NAC) of freely moving rats. Local infusion of 5-hydroxytryptamine (5-HT) or the serotonin reuptake blocker fluoxetine significantly decreased extracellular acetylcholine (ACh) in the NAC. This decrease in ACh was blocked by the 5-HT1 (and beta-adrenergic) antagonist propranolol. To test suggests that 5-HT inhibits ACh interneurons via one of the 5-HT1 receptor types. The 5HT1A agonist 8-OH-DPAT given systemically again decreased extracellular levels of ACh, and the effect was dose-dependent. The 5-HT1A effect was probably exerted in the NAC, because local infusion of 8-OH-DPAT mimicked systemic injections. These microdialysis results are similar to in vitro studies which suggest an inhibitory impact of 5-HT on ACh release in basal ganglia slices and homogenates. The decrease in extracellular ACh as measured in vivo is apparently mediated, at least in part, through a 5-HT1A receptor in the accumbens. Given the role of the NAC in behavior reinforcement, this 5-HT-ACh interaction may be involved in serotonergic treatment of depression.