Glutamatergic regulation of extracellular citrulline levels in the nucleus accumbens during an emotional conditioned reflex

Intracerebral microdialysis and high-performance liquid chromatography were used to demonstrate that the acquisition and execution of an emotional conditioned reflex in Sprague-Dawley rats are accompanied by increases in extracellular citrulline (a co-product of nitric oxide synthesis) levels in the nucleus accumbens. Injection of the selective NMDA glutamate receptor antagonist MK-801 (100 microM) into the nucleus accumbens significantly decreased the increase in extracellular citrulline seen in this structure on acquisition of the emotional conditioned reflex and completely blocked the increase induced by execution of the reflex. These data suggest that during the acquisition and execution of an emotional conditioned revels, the glutamatergic input of the nucleus accumbens acts on NMDA receptors to stimulate nitric oxide production in this part of the brain.     

NO synthase-dependent increases in extracellular citrulline levels in the nucleus accumbens in an emotional conditioned reflex

Intracerebral microdialysis/HPLC studies in Sprague-Dawley rats showed that the acquisition and execution of an emotional conditioned reflex was accompanied by an increase in the extracellular citrulline level in the nucleus accumbens; citrulline is a co-product of nitric oxide synthesis. The increase in the citrulline level evoked by execution of this reflex decreased after injection of 7-nitroindazole (0.5 mM), a selective inhibitor of neuronal NO synthase, into the nucleus accumbens, and was completely blocked by injection of N-nitroarginine (0.5 mM), a non-selective inhibitor NO synthase. The increase in the nucleus accumbens citrulline level seen during execution of the emotional conditioned reflex was prevented by administration of both of these NO synthase inhibitors. These data suggest that during the acquisition and execution of the emotional conditioned reflex, there is an increase in nitric oxide production in the nucleus accumbens, which arises predominantly as a result of activation of neuronal NO synthase. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 6, pp. 700–708, June, 2006.     

Danger-Associated Sound Signals Activate the Nitrergic System of the Medial Part of the Nucleus Accumbens

Vital microdialysis and high-performance liquid chromatography were used to show that presentation of conditioned sound signals previously combined with electrocutaneous stimulation in a conditioned reflex chamber to Sprague–Dawley rats in their home cage led to increases in the extracellular citrulline level (a co-product of nitric oxide synthesis) in the medial part of the nucleus accumbens. This increase was prevented by local administration of the neuronal NO synthase inhibitor 7-nitroindazole (0.5 mM) and was not seen in animals previously presented with sound and pain stimuli with no temporal relationship or in animals with dialysis cannulae located in the lateral part of the nucleus accumbens. These data provide evidence that danger-associated sound signals evoke activation of neuronal NO synthase in the medial part of the nucleus accumbens, leading to increases in the extracellular citrulline level and, probably, enhanced production of nitric oxide in this area of the brain.     

Effects of Blockade of Dopamine D<Subscript>2</Subscript> Receptors on Extracellular Citrulline Levels in the Nucleus Accumbens During Performance of a Conditioned Reflex Fear Response

Vital microdialysis studies on Sprague–Dawley rats using HPLC showed that performance of a conditioned reflex fear response was accompanied by an increase in the extracellular level of citrulline (a coproduct of nitric oxide synthesis) in the nucleus accumbens. Administration of the dopamine D₂ receptor antagonist raclopride (10 μM) into the nucleus accumbens decreased the magnitude of the increase in the extracellular citrulline level in this structure during performance of the conditioned reflex fear response but had no effect on its behavioral measures (the level of freezing). Doses increased investigative activity in a novel context which had been inhibited by acquisition of the conditioned reflex fear response, without affecting the investigative behavior of control animals. These data suggest that the dopaminergic input and dopamine D₂ receptors control the activity of the NO-ergic system of the nucleus accumbens during performance of the conditioned reflex fear response and may control “transfer” of fear to another behavioral situation.     

Dopamine D<Subscript>1</Subscript> Receptors Regulate the Extracellular Citrulline Level in the Nucleus Accumbens During Performance of a Conditioned Reflex Fear Reaction

Intracerebral microdialysis studies on Sprague–Dawley rats using HPLC showed that performance of a conditioned reflex fear reaction was accompanied by an increase in the extracellular citrulline (a co-product of nitric oxide synthesis) level in the nucleus accumbens. Administration of the selective D₁ receptor antagonist SCH-23390 (100 μM) into the nucleus accumbens had no long-lasting effect on the extracellular citrulline level in this structure, but reduced the magnitude of the increase in the citrulline level seen on performance of the conditioned reflex fear reaction. These data suggest that the dopaminergic input of the nucleus accumbens acts via D₁ receptors to increase NO synthase activity and nitric oxide production during performance of the conditioned reflex fear reaction. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 4, pp. 353–360, April, 2008.     

Effects of N-methyl-D-aspartate on extracellular citrulline level in the rat nucleus accumbens

In vivo microdialysis combined with high-performance liquid chromatography and electrochemical detection was used to study effects of intraaccumbal infusion of N-methyl-D-aspartate (NMDA) on the content of extracellular citrulline (a nitric oxide co-product) in the medial nucleus accumbens of Sprague-Dawley rats. The intraaccumbal NMDA infusion (10-1000 microM) dose-dependently increased the local dialysate citrulline levels (193+/-7% and 258+/-7% versus basal for the 100 and 1000 microM, respectively). The NMDA-induced increase of extracellular citrulline was completely prevented by intraaccumbal infusions through the dialysis probe both of 50 microM dizocilpine maleate (an NMDA antagonist) and of 0.5 mM N-nitro-L-arginine (a nitric oxide synthase inhibitor). Local infusion of N-nitro-L-arginine (0.5 mM) slightly decreased basal citrulline levels in the nucleus accumbens throughout the entire period of the infusion, whereas dizocilpine maleate (50 microM) had no long-lasting effect. These results suggest that NMDA receptor stimulation of the medial nucleus accumbens might cause a local nitric oxide synthase activation resulting in nitric oxide production in this brain area.     

Aspiration lesions of rat ventral hippocampus disinhibit responding in conditioned suppression or extinction, but spare latent inhibition and the partial reinforcement extinction effect.

Latent inhibition refers to a decrement in learning about a stimulus as a result of its prior non-reinforced presentation. There is evidence that lesions of nucleus accumbens and conventional hippocampal lesions both disrupt the development of latent inhibition. The partial reinforcement extinction effect reflects the observation that resistance to extinction is normally greater in animals that have been rewarded on a 50% random proportion of acquisition trials than in those rewarded on every trial. Conventional hippocampal lesions, excitotoxic lesions of hippocampus plus subiculum, or conventional lesions of nucleus accumbens abolish this effect. The present experiments examined the possibility that a projection originating in the ventral [temporal in the nomenclature proposed by Blackstad: (1956) J. comp. Neurol. 105, 417-537] subiculum and terminating in nucleus accumbens underlies the normal development of latent inhibition and the partial reinforcement extinction effect, by evaluating the performance on these two behaviours of rats with aspiration lesions in the ventral hippocampal region. There was equally clear evidence of latent inhibition and of a partial reinforcement extinction effect in controls and in rats with ventral hippocampal damage. However, superimposed on this, the hippocampal lesion induced a loss of behavioural inhibition in both paradigms. Subsequent anatomical analyses indicated that cell bodies in nearby retrohippocampal cortex had maintained intact projections to nucleus accumbens. We suggest that these extra-hippocampal projections may underlie the ability to learn to ignore irrelevant stimuli.     

Citrulline in the rat brain: immunohistochemistry and coexistence with NADPH-diaphorase.

The presence in the brain of the urea cycle intermediate citrulline in the absence of a complete urea cycle has never been adequately explained. In an attempt to clarify this problem, we developed antibodies to citrulline and determined the distribution of citrulline-immunoreactivity in fixed sections of rat brain using immunoperoxidase and indirect immunofluorescence techniques. Citrulline-positive neurons were found to have a restricted distribution within the brain. A few cells were present in the cortex and corpus callosum. A large population of strongly stained cells was diffusely scattered throughout the striatum, nucleus accumbens and olfactory tubercle. Less strongly stained cells were detected in the supraoptic and paraventricular nuclei of the hypothalamus, the dorsal raphe, and the laterodorsal and pedunculopontine tegmental nuclei of the pons. The citrulline-immunoreactive cells were similar to those previously shown to contain NADPH-diaphorase activity, and double staining experiments indicated that citrulline-immunoreactivity was present in a subpopulation of NADPH-diaphorase-positive neurons. We have recently identified NADPH-diaphorase as a nitric oxide synthase. Thus the presence of citrulline in these cells suggests that it is formed within the brain as a coproduct during nitric oxide formation from arginine.     

Activity of Neurons in the Pedunculopontine Nucleus during a Food-Related Operant Conditioned Reflex

The activity of 91 neurons in the compact and diffuse zones of the pedunculopontine nucleus of freely mobile rabbits was studied during performance of a food-related operant conditioned reflex. A total of 37.4% of the neurons recorded showed reactivity to the conditioned stimulus, which is evidence that the pedunculopontine nucleus is involved in operant learning. A significant predominance of excitatory responses to the conditioned stimulus and to food reinforcement was demonstrated. The main patterns of cell responses were identified, these reflecting the nature of the effect of the conditioned stimulus on neuron activity, the structure of the behavioral act, and the properties of the reinforcement, and demonstrating a relationship between the pedunculopontine nucleus and the processes of attention, motor learning, and reinforcement. Differences were seen in the associative reactive properties of the compact and diffuse zones of the pedunculopontine nucleus to the conditioned stimulus and reinforcement, which is evidence for the functional diversity of this formation and suggests a leading role for the cholinergic compact zone in food-related operant learning and reinforcement, the diffuse zone having a leading role in food-related classical conditioned-reflex learning.     

Electrolytic lesions of the nucleus accumbens in rats which abolish the PREE enhance the locomotor response to amphetamine

Summary The partial reinforcement extinction effect (PREE) refers to the increased resistance to extinction observed in animals trained on a partial reinforcement (PR) schedule compared with those trained on a schedule of continuous reinforcement (CR). It has been suggested that the PREE is dependent upon the integrity of the septo-hippocampal system, but recent evidence has indicated that the role originally proposed for the lateral septal nucleus may in fact be subserved by the nucleus accumbens. Experiment I therefore tested the effects of electrolytic lesions of the nucleus accumbens on the PREE. These lesions abolished the PREE, the abolition resulting from a decreased rate of extinction in the lesion CR rats coupled with an increased rate of extinction in the PR rats. These results clearly implicate the nucleus accumbens in the development of the PREE, and suggest that theoretical models of the PREE based simply upon consideration of septohippocampal interactions need radical revision. The lesion also enhanced running speeds in acquisition in both the CR and the PR groups. Experiment II therefore assessed spontaneous locomotor activity and the locomotor response to amphetamine challenge at two doses. The lesion produced no increase in spontaneous locomotion; an enhanced increase in response to 1 mg/kg amphetamine; and no changes in the stereotyped behaviours induced by 10 mg/kg amphetamine.     

Increases in glutamate release in the nucleus accumbens in rats with lesions to the hippocampal formation during an emotional conditioned response

Intracerebral dialysis was used in living animals, along with high-performance liquid chromatography with electrochemical detection, to study glutamate release into the intercellular space of the nucleus accumbens during an emotional conditioned response in rats with lesions to the hippocampal glutamatergic input to this structure. Bilateral lesioning of the hippocampal formation with ibotenic acid worsened the emotional conditioned response during its acquisition and performance, and led to increases in glutamate release into the intercellular space of the nucleus accumbens. This is the first report to demonstrate that lesions to synaptic glutamatergic transmission in the nucleus accumbens lead to compensatory increases in volume glutamatergic transmission in this area of the brain. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti, Vol. 49, No. 2, pp. 245–253, March–April, 1999.     

Contextual modulation of conditioned responses: role of the ventral subiculum and nucleus accumbens

The performance of conditioned responses (CRs) is diminished when trained subjects are tested in a novel context. This study tested the hypothesis that the flow of contextual information along the disynaptic "ESA" (entorhinal cortex-ventral subiculum-nucleus accumbens) pathway is responsible for context-related modulation of CRs. Rabbits received electrolytic or sham lesions of the ventral subiculum followed by discriminative avoidance conditioning and counterbalanced extinction sessions in the original training context, a novel context, and the original training context with a novel cue. Neuronal activity was recorded simultaneously in the nucleus accumbens, cingulate cortex, and basolateral amygdala. The lesions did not affect the acquisition of avoidance behavior or prevent the reduction of CRs in response to a novel cue. However, the lesions did reduce CR incidence during extinction, and they did eliminate a further novel-context-induced CR reduction found in controls. In addition, lesions disrupted context-dependent neuronal responses in the nucleus accumbens but not in the cingulate cortex or amygdala. These findings are interpreted as supportive of the hypothesis that the ESA pathway mediates contextual modulation of CRs during extinction.     

Neuron Activity in the Pedunculopontine Nucleus during an Operant Conditioned Defensive Reflex

The activity of 109 neurons in the compact and diffuse zones of the pedunculopontine nucleus was studied in freely mobile rabbits during the acquisition and performance of a defensive operant conditioned reflex. A total of 47% of the neurons recorded showed responsive properties to the conditioned stimulus, which is evidence for the involvement of the pedunculopontine nucleus in operant learning. A significant predominance of excitatory conditioned reflex responses to the conditioned stimulus was demonstrated, showing that the nature of the influence of the pedunculopontine nucleus on projection structures during learning is mostly excitatory. The main patterns of cell responses to the conditioned stimulus were identified, these reflecting the nature of the influence of the conditioned stimulus on neuron activity, the structure of the behavioral act, and the properties of the reinforcement, suggesting a relationship between the pedunculopontine nucleus and the processes of attention, motor learning, and reinforcement. A significant decrease in the reactivity of neurons in the pedunculopontine nucleus to the conditioned stimulus as a result of specialization due to learning was demonstrated. Differences in the associative reactive properties of the compact and diffuse zones of the pedunculopontine nucleus to the conditioned stimulus were identified, which is evidence for the functional heterogeneity of this formation and suggests a leading role for the cholinergic compact zone in operant defensive behavior.     

Carbachol injections into the nucleus accumbens disrupt acquisition and expression of fear-potentiated startle and freezing in rats

The nucleus accumbens is involved in different types of emotional learning, ranging from appetitive instrumental learning to Pavlovian fear conditioning. In previous studies, we found that temporary inactivation of the nucleus accumbens blocked both the acquisition and expression of conditioned fear. This was not due to altered dopaminergic activity as we have also found that intra-nucleus accumbens infusions of the dopamine agonist amphetamine do not affect either the acquisition or the expression of conditioned fear. Therefore, in the present study we examined whether cholinergic activity in the nucleus accumbens is involved in the acquisition and expression of conditioned fear. Specifically, the effect of intra-nucleus accumbens infusions of the unselective cholinergic agonist carbachol on the acquisition and expression of conditioned fear was assessed. Across several experiments, we measured fear to visual and acoustic conditioned stimuli and to the experimental context. Further, two different measures of conditioned fear were recorded: fear potentiation of startle and freezing. Intra-nucleus accumbens carbachol infusions disrupted acquisition as well as expression of conditioned fear, regardless of the modality of the fear-eliciting stimulus or of the specific measure of conditioned fear. This disruption of conditioned fear was not simply a by-product of enhanced motor activity which also occurred after intra-nucleus accumbens carbachol infusions. Interestingly, despite the substantial effect of intra-nucleus accumbens carbachol on expression of conditioned fear, the results of the final experiment suggest that these rats extinguish similarly to control rats. Taken together, the present results indicate that acetylcholine within the nucleus accumbens is important for the learning and retrieval of conditioned fear.     

Locus coeruleus lesions and learning in the rat.

The performance of rats with bilateral lesions of the nucleus locus coeruleus (LC) was compared with that of sham-lesioned rats using 3 avoidance and 2 appetitive learning paradigms. LC lesions which significantly reduced cortical norepinephrine produced no deficits in learning one-way active or passive shock avoidance responses, nor in acquisition or extinction of a conditioned taste aversion, nor in acquisition of a bar press response for food reinforcement, but did produce impairments in running for food in an L-shaped runway. The runway deficit could not be related to motor difficulties or differences in activity between the two groups. Although locus coeruleus lesions interfere with performance in a runway task they do not produce a general impairment in learning.     

Dopamine D1 receptor-dependent regulation of extracellular citrulline level in the rat nucleus accumbens during conditioned fear response

Nucleus accumbens (N.Acc) contains a subclass of nitric oxide (NO)-generating interneurons that are presumably regulated by the dopamine input. Receptor mechanisms underlying dopamine-NO interaction in the N.Acc are poorly understood. In the current study, we used in vivo microdialysis combined with high-performance liquid chromatography to examine participation of dopamine D1 receptors in regulation of extracellular levels of citrulline (an NO co-product) in the medial N.Acc of Sprague-Dawley rats during both pharmacological challenge and a conditioned fear response. The intraaccumbal infusion of the D1 receptor agonist SKF-38393 (100-500 microM) increased dose-dependently the local dialysate citrulline levels. The SKF-38393-induced increase in extracellular citrulline was prevented by intraaccumbal infusions of 500 microM 7-nitroindazole, a neuronal NO synthase inhibitor. In behavioral microdialysis experiment, the accumbal levels of extracellular citrulline markedly increased in rats given a mild footshock paired with tone. The presentation of the tone previously paired with footshock (the conditioned fear response) produced a "conditioned" rise of extracellular citrulline levels in the N.Acc which was attenuated by intraaccumbal infusion of 100 microM SCH-23390, a dopamine D1 receptor antagonist, and prevented by intraaccumbal infusion of 500 microM 7-nitroindazole. The results suggest that in the N.Acc, the dopamine D1 receptors might regulate the neuronal NO synthase activity; this dopamine-dependent mechanism seems to participate in activation of the neuronal NO synthase and probably NO formation in this brain area during the conditioned fear response.     

The Effects of Microinjection of Neurotensin into the Caudate Nucleus of the Rat Brain on the Performance and Extinction of a Conditioned Motor Reflex

The studies reported here demonstrate that microinjection of neurotensin into the caudate nucleus of the rat brain facilitated extinction of a conditioned motor reflex with reinforcement provided by drinking. Neurotensin had positive effects on the process of post-extinction inhibition in subsequent experiments. Neurotensin microinjections had no marked effect on the performance of the conditioned reflex, though it weakened the emotional tension of the animals in an open field test. It is concluded that the functional importance of neurotensin at the level of the caudate nucleus is associated less with the regulation of motor function than with optimizing the motivational-emotional state of the animal.     

Nucleus accumbens nitric oxide immunoreactive interneurons receive nitric oxide and ventral subicular afferents in rats

The nitric oxide generating neurons of the nucleus accumbens exert a powerful influence over striatal function, in addition, these nitrergic inputs are in a position to regulate the dopaminergic and glutamatergic inputs on striatal projection neurons. It was the aim of this study to establish the source of the glutamatergic drive to nitric oxide synthase interneurons of the nucleus accumbens. The nucleus accumbens nitric oxide-generating neurons receive asymmetrical, excitatory, presumably glutamatergic inputs. Possible sources of these inputs could be the limbic and cortical regions known to project to this area. To identify sources of the excitatory inputs to the nitric oxide synthase-containing interneurons of the nucleus accumbens in the rat we first examined the ultrastructural morphology of asymmetrical synaptic specializations contacting nitric oxide synthase-immunohistochemically labeled interneurons in the nucleus accumbens. Neurons were selected from different regions of the nucleus accumbens, drawn using camera lucida, processed for electron microscopic analysis, and the boutons contacting nitric oxide synthase-labeled dendrites were photographed and correlated to the drawings. Using vesicle size as the criterion the source was predicted to be either the prefrontal cortex or the ventral subiculum of the hippocampus. To examine this prediction, a further study used anterograde tracing from both the prefrontal cortex and the ventral subiculum, and nitric oxide synthase immunohistochemistry with correlated light and electron microscopy. Based on appositions by anterogradely labeled fibers, selected nitric oxide synthase-labeled neurons within the nucleus accumbens, were examined with electron microscopic analysis. With this technique we confirmed the prediction that subicular afferent boutons make synaptic contact with nitric oxide synthase interneurons, and demonstrated anatomically that nitric oxide synthase boutons make synaptic contact with the dendritic arbors of nitric oxide synthase interneurons. We suggest that the subicular input may excite the nitric oxide synthase neurons synaptically, while the nitric oxide synthase-nitric oxide synthase interactions underlie a nitric oxide signaling network which propagates hippocampal information, and expands the hippocampus's influence on 'gating' information flow across the nucleus accumbens.     

Facilitated extinction of appetitive instrumental conditioning following excitotoxic lesions of the core or the medial shell subregion of the nucleus accumbens in rats

The nucleus accumbens has been implicated in the control of goal-directed behaviour, including instrumental conditioning. Here, we evaluated the effect of N-methyl-d-aspartate (NMDA)-induced excitotoxic lesions restricted to either the core or the medial shell subregions of the nucleus accumbens (NAC) on extinction in rats using a trial discrete fixed ratio-5 (FR-5) appetitive operant procedure. Neither core nor shell lesions of the NAC affected the acquisition of instrumental responding. Both lesions facilitated the cessation of responding when the instrumental act no longer yielded reinforcement. Our results suggest that both the NAC core and medial shell contribute to the control of extinction learning of appetitively motivated instrumental behaviour.     

Immunohistochemical localization of argininosuccinate synthetase in the rat brain in relation to nitric oxide synthase-containing neurons.

The distribution of the urea cycle enzyme, argininosuccinate synthetase, in the rat brain was determined using immunohistochemistry. This enzyme participates in the only known metabolic pathway for citrulline, its condensation with aspartate to form argininosuccinate, which can then be cleaved to fumarate and arginine. It may thus provide a mechanism to recycle citrulline, formed in the nervous system via nitric oxide synthase activity, back to the nitric oxide precursor, L-arginine. Argininosuccinate synthetase immunoreactivity was detected in discrete populations of neurons throughout the brain. Double-staining with nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry for the localization of nitric oxide synthase demonstrated that argininosuccinate synthetase coexists with nitric oxide synthase in some brain regions. However, many neurons were found that contained one of these two enzymes, but not the other. Thus some nitric oxide synthase-containing neurons appear able to recycle citrulline via argininosuccinate, while others do not. Additional roles for argininosuccinate synthetase in the brain are discussed.