AMPA receptor antagonists reverse effects of extended habit training on signaled food approach responding in rats.

RATIONALE: Dopamine D1 receptor stimulation is critically involved in early appetitive phases of learning in various behavioral paradigms. However, extended habit training was previously shown to reduce the ability of dopamine D1 receptor antagonists such as R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390) to disrupt behavioral performance. OBJECTIVE: The present study aimed to evaluate whether coadministration of glutamate alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor antagonists restores sensitivity to acute blockade of D1 receptors. MATERIALS AND METHODS: Adult male Wistar rats were presented with 45-mg food pellets delivered to the food tray, which was immediately preceded by a 400-ms tone (2.8 kHz, 78 dB). During each training and test session, there were 28 food-tone presentations with an average intertrial interval of 70 s, and each head entry into the food tray was recorded. Drug tests were conducted on either day 3 or 9 of the training using independent groups of animals. The main dependent variable was the number of trials during which no head-entry response was made during the 10-s period immediately after the food delivery. RESULTS: Longer training duration enhanced the resistance of the signaled food approach behavior to extinction and to disrupting effects of supplementary food ration. Similarly, acute administration of SCH-23390 (0.04-0.16 mg/kg) dose-dependently reduced the number of omitted trials when given before the test session on day 3 but much less so when injected on day 9. AMPA receptor antagonists, NBQX (10 mg/kg) or GYKI-52466 (3-10 mg/kg), had no effects on their own but significantly enhanced the disrupting effects of SCH-23390 (0.08 and 0.16 mg/kg) when given on day 9 but not on day 3 of the training. CONCLUSIONS: These results indicate that AMPA receptor blockade restores sensitivity to appetitive behavior-disrupting effects of SCH-23390 in subjects exposed to extended training protocol.

Latent inhibition of conditioned taste aversion is not disrupted, but can be enhanced, by selective nucleus accumbens shell lesions in rats.

Latent inhibition is a form of negative priming in which repeated non-reinforced pre-exposures to a stimulus retard subsequent learning about the predictive significance of that stimulus. The nucleus accumbens shell and the anatomical projection it receives from the hippocampal formation have been attributed a pivotal role in the control or regulation of latent inhibition expression. A number of studies in rats have demonstrated the efficacy of selective shell lesions to disrupt latent inhibition in different associative learning paradigms, including conditioned active avoidance and conditioned emotional response. Here, we extended the test to the conditioned taste aversion paradigm, in which the effect of direct hippocampal damage on latent inhibition remains controversial. We demonstrated the expected effect of selective shell lesions on latent inhibition of conditioned emotional response and of conditioned active avoidance, before evaluating in a separate cohort of rats the effect of comparable selective lesions on latent inhibition of conditioned taste aversion: a null effect of the lesions was first obtained using parameters known to be sensitive to amphetamine treatment, then an enhancement of latent inhibition was revealed with a modified conditioned taste aversion procedure. Our results show that depending on the associative learning paradigm chosen, shell lesions can disrupt or enhance the expression of latent inhibition; and the pattern is reminiscent of that seen following hippocampal damage.

A differential involvement of the shell and core subterritories of the nucleus accumbens of rats in attentional processes.

The nucleus accumbens comprises of two anatomically distinct subterritories: an inner core and an outer shell region. The distinct pattern of the core and shell input and output targets suggests that these two regions may mediate different behavioral processes. Using N-methyl-D-aspartate excitotoxic lesions in either the core or shell region, we investigated whether we can dissociate functionally these two subterritories. N-Methyl-D-aspartate-lesioned, sham-lesioned and non-operated animals were tested for locomotor activity in an open field and in two behavioral paradigms known to evaluate attentional deficits, namely the pre-pulse inhibition of the acoustic startle reflex and latent inhibition, measured in a two-way active avoidance paradigm. The shell-lesioned animals showed a small but significant hyperactivity in the open field when compared to the core-lesioned and to control animals. In the pre-pulse inhibition paradigm, core-lesioned animals demonstrated reduced pre-pulse inhibition to the two high pre-pulse intensities (80 dB[A], 84 dB[A]). In the active avoidance paradigm, whereas no lesion effects were detected in the non-pre-exposed groups, clear attenuation of latent inhibition was found in the shell-lesioned rats, in comparison to both core-lesioned and control rats, due to improved avoidance performance of the shell-pre-exposed group. From these results we suggest that the two subterritories of the nucleus accumbens are differentially involved in attention-related processes: the core lesion leads to significant disruption of pre-pulse inhibition while the shell lesion leads to heightened activity and significant attenuation of latent inhibition.

A double labeling technique using WGA-apoHRP-gold as a retrograde tracer and non-isotopic in situ hybridization histochemistry for the detection of mRNA.

We describe a novel method to study the neurochemical nature of a specific neuronal pathway by using conjugated WGA-apoHRP as a retrograde tracer and non-isotopic in situ hybridization histochemistry to examine the expression of mRNA. The technique was developed to eliminate the reduction of retrograde tracer during the rigorous procedures involved in in situ hybridization. The tracer was injected stereotaxically into the brainstem of Macaca fascicularis monkeys. Sections through the central nucleus of the amygdala were processed for the visualization of the retrogradely transported WGA-apoHRP-gold using a silver enhanced reaction, followed by non radioactive in situ hybridization for the mRNA encoding glutamic acid decarboxylase (GAD67). Numerous retrogradely labeled cells were observed in the central nucleus of the amygdala. Comparison of double-labeled sections with sections processed for the retrograde tracer alone indicated that there was relatively little loss of the retrograde tracer during the in situ hybridization processing. This method provides a relatively simple and reliable tool to study the molecular phenotype of identified projection neurons.

Testing the disinhibition hypothesis of epileptogenesis in vivo and during spontaneous seizures.

The "disinhibition" hypothesis contends that (1) seizures begin when granule cells in the dentate gyrus of the dorsal hippocampus are disinhibited and (2) disinhibition occurs because GABAergic interneurons are excessively inhibited by other GABAergic interneurons. We tested the disinhibition hypothesis using the experimental model that inspired it-naturally epileptic Mongolian gerbils. To determine whether there is an excess of GABAergic interneurons in the dentate gyrus of epileptic gerbils, as had been reported previously, GABA immunocytochemistry, in situ hybridization of GAD67 mRNA, and the optical fractionator method were used. There were no significant differences in the numbers of GABAergic interneurons. To determine whether granule cells in epileptic gerbils were disinhibited during the interictal period, IPSPs were recorded in vivo with hippocampal circuits intact in urethane-anesthetized gerbils. The reversal potentials and conductances of IPSPs in granule cells in epileptic versus control gerbils were similar. To determine whether the level of inhibitory control in the dentate gyrus transiently decreases before seizure onset, field potential responses to paired-pulse perforant path stimulation were obtained from the dorsal hippocampus while epileptic gerbils experienced spontaneous seizures. Evidence of reduced inhibition was found after, but not before, seizure onset, indicating that seizures are not triggered by disinhibition in this region. However, seizure-induced depression of inhibition may amplify and promote the spread of seizure activity to other brain regions. These findings do not support the disinhibition hypothesis and suggest that in this model of epilepsy seizures initiate by another mechanism or at a different site.

Disruption of prepulse inhibition following N-methyl-D-aspartate infusion into the ventral hippocampus is antagonized by clozapine but not by haloperidol: a possible model for the screening of atypical antipsychotics.

The present study tested the effects of the typical neuroleptic haloperidol and an atypical neuroleptic clozapine on ventral hippocampus stimulation-induced disruption of prepulse inhibition (PPI). Bilateral infusions of 0.7 microg NMDA into the ventral hippocampus disrupted PPI. The impairment of PPI following the infusion was completely normalized 24 h after the infusion. This disruption of PPI was antagonized by clozapine (5.0 mg/kg), but not by haloperidol (0.2 mg/kg). Since disruption of PPI is considered to constitute an animal model of schizophrenia that is related to the deficit of sensorimotor gating observed in schizophrenic patients, these results suggest that PPI disruption induced by intra-ventral hippocampal infusions of NMDA may serve as an animal model for the selective detection of atypical antipsychotics.

Highly specific neuron loss preserves lateral inhibitory circuits in the dentate gyrus of kainate-induced epileptic rats.

Patients with temporal lobe epilepsy display neuron loss in the hilus of the dentate gyrus. This has been proposed to be epileptogenic by a variety of different mechanisms. The present study examines the specificity and extent of neuron loss in the dentate gyrus of kainate-treated rats, a model of temporal lobe epilepsy. Kainate-treated rats lose an average of 52% of their GAD-negative hilar neurons (putative mossy cells) and 13% of their GAD-positive cells (GABAergic interneurons) in the dentate gyrus. Interneuron loss is remarkably specific; 83% of the missing GAD-positive neurons are somatostatin-immunoreactive. Of the total neuron loss in the hilus, 97% is attributed to two cell types-mossy cells and somatostatinergic interneurons. The retrograde tracer wheat germ agglutinin (WGA)-apoHRP-gold was used to identify neurons with appropriate axon projections for generating lateral inhibition. Previously, it was shown that lateral inhibition between regions separated by 1 mm persists in the dentate gyrus of kainate-treated rats with hilar neuron loss. Retrogradely labeled GABAergic interneurons are found consistently in sections extending 1 mm septotemporally from the tracer injection site in control and kainate-treated rats. Retrogradely labeled putative mossy cells are found up to 4 mm from the injection site, but kainate-treated rats have fewer than controls, and in several kainate-treated rats virtually all of these cells are missing. These findings support hypotheses of temporal lobe epileptogenesis that involve mossy cell and somatostatinergic neuron loss and suggest that lateral inhibition in the dentate gyrus does not require mossy cells but, instead, may be generated directly by GABAergic interneurons.

Distribution of GABAergic cells and fibers in the hippocampal formation of the macaque monkey: an immunohistochemical and in situ hybridization study.

The gamma-aminobutyric acid (GABAergic) system of the hippocampal formation of Macaca fascicularis monkeys was studied immunohistochemically with a monoclonal antibody to GABA and with nonisotopic in situ hybridization with cRNA probes for glutamic acid decarboxylase 65 (GAD65) and GAD67. The highest densities of labeled cells were observed in the presubiculum, parasubiculum, entorhinal cortex, and subiculum, whereas the CA3 field and the dentate gyrus had the lowest densities of positive neurons. Within the dentate gyrus, most of the GABAergic neurons were located in the polymorphic layer and in the deep portion of the granule cell layer. GABAergic terminals were densest in the outer two-thirds of the molecular layer. GABAergic neurons were seen throughout all layers of the hippocampus. Terminal labeling was highest in the stratum lacunosum-moleculare. A higher terminal labeling was observed in the subiculum than in CA1 and was particularly prominent in layer II of the presubiculum. A bundle of GABAergic fibers was visible deep to the cell layers of the presubiculum and subiculum. This bundle could be followed into the angular bundle ipsilaterally and was continuous with stained fibers in the dorsal hippocampal commissure. This pattern of labeling is reminiscent of the presubicular projections to the contralateral entorhinal cortex. GABAergic cells were observed in all layers of the entorhinal cortex although the density was higher in layers II and III than in layers V and VI. The in situ hybridization preparations largely confirmed the distribution of GABAergic neurons in all fields of the hippocampal formation.

Evidence for a GABAergic projection from the central nucleus of the amygdala to the brainstem of the macaque monkey: a combined retrograde tracing and in situ hybridization study.

The central nucleus of the amygdala is interconnected with a variety of visceral and autonomic nuclei of the brainstem. These include the parabrachial nucleus, the nucleus of the solitary tract, the nucleus ambiguus and the dorsal motor nucleus of the vagus. Despite repeated attempts, neurochemical characterization of the major subcortical connections of the central nucleus has not yet been accomplished. Based on earlier immunohistochemical and in situ hybridization evidence indicating the presence of numerous GABAergic neurons in the macaque monkey central nucleus, we predicted that a sizeable portion of the descending projections may be GABAergic. We tested this hypothesis using a novel double labelling method with gold conjugated WGA-apoHRP as a retrograde tracer and in situ hybridization for detecting the mRNA that encodes the enzyme glutamic acid decarboxylase (GAD67) as a marker for GABAergic cells. Following WGA-apoHRP-gold injections into the brainstem, a large number of retrogradely labelled cells was observed in the medial and lateral divisions of the central nucleus. Of the retrogradely labelled cells observed in the medial division of the central nucleus, approximately half were double-labelled for GAD67 mRNA; about 30% double labelling was observed in the lateral division. These data support the view that a sizeable component of the central nucleus projection to the brainstem is GABAergic.

Differential localization of mRNAs encoding dopamine D1 or D2 receptors in cholinergic neurons in the core and shell of the rat nucleus accumbens.

By combining immunocytochemistry for ChAT and in situ hybridization for dopamine D1 or D2 receptor mRNA in the striatum, it was found that (1) the percentage of ChAT/D2 mRNA co-localization is higher in the caudate-putamen than in the shell and core of the nucleus accumbens, (2) in the shell the degree of ChAT/D2 mRNA co-localization is higher rostrally than caudally, and 3) no significant regional differences exist in the degree of co-localization of ChAT and D1 mRNA.

Differential effects of dopamine depletion on the binding and mRNA levels of dopamine receptors in the shell and core of the rat nucleus accumbens.

In the present study, using quantitative receptor autoradiography and in situ hybridization histochemistry the effects of unilateral 6-hydroxydopamine lesions on the binding density levels of dopamine D1 and D2 receptors and the levels of mRNA encoding D1 and D2 receptors were investigated in the core and shell territories of the nucleus accumbens (Acb) and in the caudate-putamen (CP). The lesions induced contrasting effects on the D1 binding and D1 mRNA in the Acb and CP, i.e. an increase in binding and a decrease in the mRNA levels. For the D2 receptor an increase in both the binding density and mRNA levels was observed. The lesion-induced effects displayed regional differences. For D1 mRNA and D1 and D2 binding, the lesion effect was more pronounced in the core than in the shell of the Acb. For the D2 mRNA levels an increase was observed in the CP but not in the two territories of the Acb. Furthermore, the decrease in D1 mRNA was greater in the rostral than in the caudal parts of the core and shell of the Acb. These results indicate that the core and shell of the Acb and the CP respond differentially to dopamine depletion.

Immunohistochemical characterization of the shell and core territories of the nucleus accumbens in the rat.

The nucleus accumbens in the rat has been parcelled into shell and core subdivisions. Despite accumulating evidence for such a division of the nucleus accumbens, these territories have not been delineated throughout the rostrocaudal extent of the nucleus. In the present study, an attempt has been made to delineate the shell and core using the distribution of calcium-binding protein immunoreactivity, substance P immunoreactivity and acetylcholinesterase activity in transverse and horizontal sections through the nucleus accumbens. It was found that the pattern of calcium-binding protein immunoreactivity provides the most unequivocal criterion to divide the nucleus accumbens into a ventral and medial, peripheral shell displaying low to moderate immunostaining, and a more laterally and dorsally located, strongly stained inner core. In most parts of the nucleus, borders seen in the calcium-binding protein immunoreactivity pattern can also be recognized in the distributions of substance P immunoreactivity and acetylcholinesterase activity. It is concluded that the shell occupies most of the rostral part of the nucleus accumbens, whereas rostrally the core is represented only in the most lateral part. Differences in staining intensities for all three markers indicate that both the shell and core have a heterogeneous structure. Patterns of connectivity appear to support the division of the nucleus accumbens as indicated by calcium-binding protein immunoreactivity in the present study.

Rostrocaudal subregional differences in the response of enkephalin, dynorphin and substance P synthesis in rat nucleus accumbens to dopamine depletion.

Quantitative in situ hybridization histochemistry was used to examine the effects of unilateral 6-hydroxydopamine lesions of the ascending dopaminergic fibres on levels of mRNA encoding the neuropeptides enkephalin, dynorphin and substance P in subregions of the nucleus accumbens. The nucleus accumbens was divided into quadrants and changes in mRNA were measured along the rostrocaudal extent of the nucleus. Two weeks after the lesion an increase was found in enkephalin mRNA in the lesioned side compared to the non-lesioned side, whereas a decrease was observed for dynorphin and substance P mRNA. The changes in mRNA levels differed from quadrant to quadrant and were not uniformly distributed along the rostrocaudal axis. Both types of changes, i.e. increase and decrease, were much higher in rostral parts of the nucleus than in caudal parts, indicating regional differences in the effects of blockade of the dopaminergic neurotransmission. The lesion-induced increases and decreases in mRNA levels occurred in both the shell and the core subregions of the nucleus accumbens and were not specifically related to either of these areas. Factors are discussed that may contribute to the rostrocaudal gradient in the changes of enkephalin, substance P and dynorphin mRNA levels. On the basis of their afferent and efferent connections, the rostral and caudal parts of the nucleus accumbens are considered to be involved in different functions. The present results suggest that dopamine depletion may affect these functions in a differential manner.

Evidence for a multi-compartmental histochemical organization of the nucleus accumbens in the rat.

In the present study, the compartmental organization of the nucleus accumbens was investigated by comparing the pattern of leu-enkephalin immunoreactivity with that of the opioid receptor ligand, naloxone, an established marker for the compartmental organization of the neostriatum. Both patterns have a nonhomogeneous, patch-like appearance throughout the rostrocaudal extent of the nucleus and show a good, mutual correspondence. In the core of the nucleus accumbens as well as in the border region between the nucleus accumbens and the caudate-putamen, leu-enkephalin-rich areas are in register with opioid receptor-dense areas. In the shell region the precise relationship between the enkephalin and the naloxone patterns could not be established. A comparison of the connectivity patterns and neurochemical characteristics of the opioid receptor-dense compartments in the nucleus accumbens with those in the caudate-putamen reveals major discrepancies between these two striatal subdivisions. We therefore conclude that, rather than a bicompartmental patch/striosome-matrix organization, the nucleus accumbens has a multicompartmental organization.

Role of neostriatum and nucleus accumbens in stepping induced by apomorphine and dexamphetamine.

Systemic administration of apomorphine and dexamphetamine are known to produce circling in rats by changing the functioning of hindlimb stepping and forelimb stepping, respectively. In the present study intracranial injections of the dopamine antagonist sulpiride were used to study the involvement of the neostriatum and nucleus accumbens in these effects. It was found that injections of sulpiride into the neostriatum, but not the nucleus accumbens, dose-dependently (1-20 ng) reduced the number of apomorphine-induced hindlimb doublets without affecting the dexamphetamine-induced forelimb crossing steps. On the other hand, it was found that injections into the nucleus accumbens, but not the neostriatum, reduced the number of dexamphetamine-induced forelimb crossing steps without affecting the apomorphine-induced hindlimb doublets. This effect was not dose-dependent. The data suggest that the neostriatum, but not the nucleus accumbens, is primarily involved in the apomorphine-induced changes in hindlimb stepping, and that the nucleus accumbens, but not the neostriatum, is primarily involved in the dexamphetamine-induced changes in forelimb stepping.