The role of D2 dopamine receptors in oxytocin induced place preference and anxiolytic effect.

Neuropeptide oxytocin (OT) is involved in the regulation of social and non-social behaviour. The central nucleus of amygdala (CeA), part of the limbic system, plays an important role in learning, memory, anxiety and reinforcing mechanisms. CeA has been shown to be rich in OT receptors in rodents. Our previous findings indicated that OT in the rat CeA has a dose dependent rewarding and anxiolytic effect. The aim of our present study was to examine in the CeA the possible interaction of OT and D2 dopamine (DA) receptor antagonist Sulpiride on reinforcement in place preference test and on anxiety in elevated plus maze test. Wistar rats were microinjected bilaterally with 10 ng OT. In different group of animals 4 µg D2 DA receptor antagonist was applied. Other animals received D2 DA receptor antagonist 15 min before 10 ng OT treatment or vehicle solution into the CeA. Rats receiving 10 ng OT spent significantly longer time in the treatment quadrant during the test session in conditioned place preference test. Prior treatment with D2 DA receptor antagonist blocked the rewarding effects of OT. Antagonist in itself did not influence the time rats spent in the treatment quadrant. In elevated plus maze test, rats receiving 10 ng OT spent significantly longer time on the open arms. Prior treatment with D2 DA receptor antagonist blocked the effects of OT. Our results show that DA system plays a role in positive reinforcing and anxiolytic effects of OT because D2 DA receptor antagonist can block these actions.

The role of intraamygdaloid neurotensin and dopamine interaction in conditioned place preference.

Tridecapeptide Neurotensin (NT) is widely distributed in the central nervous system where it acts as a neurotransmitter and neuromodulator. The central nucleus of amygdala (CeA), part of the limbic system, plays an important role in learning, memory, anxiety and reinforcing mechanisms. Our previous data showed that NT microinjected into the CeA has positive reinforcing properties. We supposed that these effects might be due to modulations of the mesolimbic dopamine system. The aim of our study was to examine in the CeA the possible effects of NT and dopamine interaction on reinforcement by conditioned place preference test. Male Wistar rats were microinjected bilaterally with 100 ng NT or 2 µg D1 dopamine receptor antagonist alone, or D1 dopamine antagonist 15 min before 100 ng NT treatment or vehicle solution into the CeA. Other animals received 4 µg D2 dopamine receptor antagonist Sulpiride alone, or administration of D2 dopamine receptor antagonist 15 min before 100 ng NT treatment or vehicle solution into the CeA. Rats that received 100 ng NT spent significantly more time in the treatment quadrant during the test session. Pre-treatment with the D1 dopamine antagonist, blocked the effects of NT. D2 dopamine receptor antagonist pretreatment could prevent the positive reinforcing effects of NT as well. Antagonists themselves did not influence the place preference. Our results show that the rewarding effect of NT can be due to the modulation of DA system, since its effects could be blocked by either D1 dopamine or D2 dopamine antagonist preteatment.

Substance P and neurotensin in the limbic system: Their roles in reinforcement and memory consolidation.

Substance P (SP) and neurotensin (NT) are neuropeptides isolated in the periphery and in the central nervous system. They are involved in various regulatory processes in the gastrointestinal tract, in the circulatory and respiratory systems, kidney and endocrine system. In addition to the peripheral effects, SP and NT act as neurotransmitters and neuromodulators in the central nervous system, regulating various behavioural actions, such as general and motor activity, pain, food and water intake, anxiety, reward/reinforcement and memory consolidation. In the limbic system SPergic and NTergic pathways, terminals and related receptors have been identified. According to several data of literature and to our recently published results, SP and NT have rewarding/reinforcing effects and facilitate memory consolidation in various limbic regions. In this report evidences are provided about the interaction of these neuropeptides with dopaminergic and acetylcholinergic systems. A hypothesis is presented that rewarding/reinforcing effects of SP and NT develop by modulating the mesencephalic dopaminergic system, while their mnemonic effects are mediated via the mesencephalic dopaminergic and the basal forebrain cholinergic systems.

Food and water intake, body temperature and metabolic consequences of interleukin-1ß microinjection into the cingulate cortex of the rat.

In order to elucidate whether cytokine mechanisms of the cingulate cortex (cctx) are important in the central regulation of homeostasis, in the present study, feeding-metabolic effects of direct bilateral microinjection of interleukin-1ß (IL-1ß) into the cctx of the rat have been investigated. Short- (2h), medium (12h) and long-term (24h) food and water intakes and body temperature were measured after the intracerebral administration of this primary cytokine or vehicle solution, with or without paracetamol pretreatment. The effect of IL-1ß on the blood glucose level of animals was examined in glucose tolerance test (GTT), and concentrations of relevant plasma metabolites (total cholesterol, HDL, LDH, triglycerides, uric acid) were additionally also determined following the above microinjections. In contrast to causing no major alteration in the food and water intakes, the cytokine treatment evoked significant increase in the body temperature of the rats. Prostaglandin-mediated mechanisms were shown to have important role in the mode of this action of IL-1ß, since paracetamol pretreatment partially prevented the development of the above mentioned hyperthermia. In the GTT, no considerable difference was observed between the blood glucose levels of the cytokine treated and control animals. Following IL-1ß microinjection, however, significant decrease of HDL and total cholesterol was found. Our present findings indicate that elucidating the IL-1ß mediated homeostatic control mechanisms in the cingulate cortex may lead to the better understanding not only the regulatory entities of the healthy organism but also those found in obesity, diabetes mellitus and other worldwide rapidly spreading feeding-metabolic disorders.

Impaired glucose tolerance after streptozotocin microinjection into the mediodorsal prefrontal cortex of the rat.

The mediodorsal prefrontal cortex (mdPFC) is a key structure of the central glucose-monitoring (GM) neural network. Previous studies indicate that intracerebral streptozotocin (STZ) microinjection-induced destruction of local chemosensory neurons results in feeding and metabolic alterations. The present experiments aimed to examine whether STZ microinjection into the mdPFC causes metabolic deficits. To do so, glucose tolerance test (GTT) and measurements of plasma metabolites were performed in STZ-treated or control rats. Intraperitoneal D-glucose load was delivered 20 min or 4 weeks following the intracerebral microinjection of STZ or saline (acute or subacute GTT, respectively). The STZ-treated rats displayed acute glucose intolerance: at the 120th min of the test, blood glucose level of these rats was significantly higher than that of the ones in the control group. When determining the plasma level of various metabolites, 30 min following the intracerebral STZ or saline microinjection, the triglyceride concentration of the STZ-treated rats was found to be reduced compared with that of the control rats. The GM neurons of the mdPFC are suggested to be involved in the organization of complex metabolic processes by which these chemosensory cells contribute to adaptive control mechanisms of the maintenance of homeostasis.

Insulin and leptin plasma levels after the microinjection of interleukin-1ß into the nucleus accumbens of the rat.

The nucleus accumbens (NAcc), an important basal forebrain structure, has a central integratory function in the control of feeding and metabolism. The primary cytokine interleukin-1ß (IL-1ß) exerts its neuromodulatory effects on the endocrine functions both centrally and peripherally. The present study was designed to elucidate the possible consequences of direct administration of IL-1ß into the NAcc on the endocrine regulation of metabolism. Plasma concentrations of insulin and leptin, two key hormones in the homeostatic control were determined 15 minutes after a single bilateral microinjection of IL-1ß into the NAcc of adult male Wistar rats, and the effects were compared with those found in vehicle treated control animals. Insulin plasma levels of the cytokine treated animals were significantly higher than those parameters of the control rats. No differences were found in leptin plasma concentrations between the two groups. Our findings show that IL-1ß mediated processes in the NAcc have important roles in the central neuroendocrine control.

Regulatory processes of hunger motivated behavior.

While food intake and body weight are under homeostatic regulation, eating is a highly motivated and reinforced behavior that induces feelings of gratification and pleasure. The chemical senses (taste and odor) and their evaluation are essential to these functions. Brainstem and limbic glucose-monitoring (GM) neurons receiving neurochemical information from the periphery and from the local brain milieu are important controlling hunger motivation, and brain gut peptides have a modulatory role on this function. The hypothalamic and limbic forebrain areas are responsible for evaluation of reward quality and related emotions. They are innervated by the mesolimbic dopaminergic system (MLDS) and majority of GM neurons are also influenced by dopamine. Via dopamine release, the MLDS plays an essential role in rewarding-reinforcing processes of feeding and addiction. The GM network and the MLDS in the limbic system represent essential elements in the neural substrate of motivation.

Alterations of conditioned taste aversion after microiontophoretically applied neurotoxins in the medial prefrontal cortex of the rat.

The prefrontal cortex (PFC) has been reported to be essential in neural control of feeding. In the present study, we aimed to provide a complex characterization of behavioral consequences of PFC microlesions in CFY rats. Kainic acid (KA) was microiontophoretically applied into the mediodorsal division of PFC to damage intrinsic neurons, whereas in another group of rats, 6-hydroxydopamine (6-OHDA) was microiontophoretized into the same region to destroy catecholaminergic (CA) projection fiber terminals. Body weights, food and fluid intake of both lesioned and (sham-operated or intact) control animals were daily measured. Effects of intracellular dehydration and water deprivation were also studied. Open field activity, stereotyped behaviors, and orientation towards visual and somesthetic stimuli were pre- and postoperatively tested. To examine hypothesized consequences of mPFC microlesions on central taste information processing, the acquisition and retention of saccharine conditioned taste aversion (CTA) were studied. No major changes were recorded in body weights, food and water consumption. Dehydration or deprivation similarly increased water intake in all animals. Scores of open field activity and stereotyped behaviors in the 6-OHDA group were significantly higher than those of the other groups. As the main findings of the present studies, both KA and 6-OHDA lesioned rats displayed significant deficits in CTA acquisition and retention tests. These results suggest that the medial PFC has a substantial role in both the formation and the retrieval of CTA. Furthermore, the present findings also indicate the general significance of prefrontal CA mechanisms in the organization of goal-directed, adaptive behaviors.

Complex functional attributes of amygdaloid gustatory neurons in the rhesus monkey.

To reveal specific functions of glucose-sensitive (GS) and glucose-insensitive (GIS) cells in chemical information processing, single neuron activity was recorded in the amygdaloid body (AMY) of macaques during: 1) gustatory stimulations and 2) micro-electrophoretic administration of chemicals. Of the 629 neurons tested, 56 (8.9%) responded to, usually two or more, taste qualities. Hedonically distinct tastants usually elicited opposite firing rate changes of the gustatory cells. Seventy percent of the gustatory responses were recorded from GS neurons (17% of all AMY cells). Catecholamines (CAs) induced discharge rate changes in a majority of taste-responsive neurons: The GS gustatory cells were suppressed by norepinephrine (in the form of noradrenaline HCl, NA), whereas the GIS taste-responsive neurons were facilitated by dopamine (DA). Furthermore, NA- and/or DA-antagonists were able to attenuate or suppress taste-elicited responses of several of these cells. These and previous data indicate a specific functional organization of AMY gustatory cells: The GS and GIS taste neurons appear to be involved in differential integration of feeding-associated humoral-metabolic, motivational and exogenous chemical information.

Facilitation of glutamate release in the ventromedial division of the globus pallidus during palatable taste stimulation in freely moving rats: real-time measurement.

To make real-time measurements of glutamate in the ventromedial globus pallidus (vGP) in rats during free ingestive behavior, a recently developed dialysis biosensor was employed. The glutamate level in the vGP increased in response to intraoral infusions of various fluids and voluntary ingestion of food pellets. Palatable fluids evoked greater responses than unpalatable fluids did, suggesting that glutamate in the vGP is involved in ingestive behavior.

Differential roles of monkey striatum in learning of sequential hand movement.

To study the role of the basal ganglia in learning of sequential movements, we trained two monkeys to perform a sequential button-press task (2x5 task). This task enabled us to examine the process of learning new sequences as well as the execution of well-learned sequences repeatedly. We injected muscimol (a GABA agonist) into different parts of the striatum to inactivate the local neural activity reversibly. The learning of new sequences became deficient after injections in the anterior caudate and putamen, but not the middle-posterior putamen. The execution of well-learned sequences was disrupted after injections in the middle-posterior putamen and, less severely, after injections in the anterior caudate/putamen. These results suggest that the anterior and posterior portions of the striatum participate in different aspects of learning of sequential movements.

Disturbances of neophobia and taste-aversion learning after bilateral kainate microlesions in the rat pallidum.

These experiments aimed to elucidate feeding-associated behavioral roles of globus pallidus (GP) neurons in gustatory functions: The effects of bilateral microiontophoretic kainate (KA) lesions of the ventromedial pallidal (vmGP) region on neophobia and conditioned taste aversion (CTA) were studied. Lesioned rats displayed strong and persistent neophobia to a mild citric acid solution. Neuron-specific damage to the vmGP also prevented rats from proper acquisition of CTA. Rats that previously showed normal neophobia and successfully learned CTA demonstrated difficulties in CTA retention after GP lesions. KA-lesioned rats, in addition, exhibited deficits in orientation reactions but did not have aphagia, adipsia, or motor disturbances seen after larger pallidal lesions. These findings suggest that neurons of the GP are significant in acquisition, memory storage, and retrieval mechanisms of feeding-associated taste information.

Distribution and time course of appearance of "dark" neurons and EEG activity after amygdaloid kainate lesion.

To determine the extent and time course of local and distant neuronal damage produced by microiontophoretic administration of kainic acid (KA) into the central amygdaloid nucleus, distribution of neuronal damage was compared in various brain areas after different survival times. For demonstration of damaged, so-called "dark" neurons, a newly developed silver stain was employed. In addition, silver staining method was used to visualize microglia cells. In a separate experiment, electroencephalographic (EEG) activity was recorded from the amygdaloid body, hippocampus, and the frontal cortex before and after microiontophoretic KA lesion of the central amygdaloid nucleus. It was observed that (1) even a minute amount of KA into this nucleus caused transient neuronal damage in distant brain areas; (2) the hippocampal formation, subiculum, entorhinal cortex, piriform cortex, and lateral septum were consistently affected; (3) the extent and time course of neuronal damage and appearance of microglia cells varied from area to area; (4) the KA neurotoxicity in distant brain areas appeared to depend on specific excitatory circuits, especially in the hippocampal formation; (5) the appearance and time course of pathologic EEG activity paralleled the appearance of dark neurons; and (6) the absence of pathologic EEG activity and the lack of massive neuronal loss or microglia proliferation in distant brain areas of rats surviving longer than 48 h suggested that these areas may have recovered both morphologically and functionally. Although details of cellular mechanism responsible for development of "dark" degeneration of neurons are not known, the silver method employed in the present study proved to be sensitive, useful tool for fine histological analyses of early and distant consequences of excitotoxic lesions.

Responses of forebrain neurons to the MAO-B blocker L-deprenyl.

Despite the large amount of neuropharmacological data concerning catecholamine (CA) mechanisms of the mammalian brain, little is known yet about the effects of MAO-inhibitors on single neurons. The present series of experiments aim to elucidate these specific neurochemical attributes of forebrain cells. Single neuron activity was recorded by means of multi-barreled microelectrodes in the caudate nucleus, globus pallidus, and amygdala of both anesthetized rats and anesthetized or alert monkeys during microelectrophoretic application of the MAO-B blocker L-deprenyl (DEPR). CAs (dopamine and noradrenaline), glutamate, GABA, and acetylcholine were also applied. Nearly the half (46%) of all forebrain neurons tested responded, exclusively with inhibition, to DEPR, and the CA-sensitive cells were especially responsive to the MAO-B inhibitor. The time course of DEPR-induced neuronal suppression was short. In some cases, amphetamine (AMPH) and clorgyline (CLOR) were also applied microelectrophoretically. AMPH elicited similar activity changes to those seen after DEPR administrations, whereas CLOR applications were less effective. Our results provide evidence that DEPR can effectively modulate the activity of CA-sensitive neurons in the three different forebrain regions of two different species. On the basis of this data, the possible neurochemical mechanisms of DEPR action are discussed.

Role of forebrain glucose-monitoring neurons in the central control of feeding: II. Complex functional attributes.

Our parallel investigations in the lateral hypothalamic are (LHA), amygdaloid body (AMY) and globus pallidus (GP) provided evidence for the existence of glucose-sensitive (GS) neurons in these forebrain regions. To examine exogenous chemosensory responsiveness of these cells, extracellular single neuron activity was recorded in anesthetized or alert rhesus monkeys and in anesthetized rats during 1) microelectrophoretic administration of chemicals and 2) gustatory and 3) olfactory stimulations. The GS cells in all three forebrain structures were more likely than the glucose-insensitive (GIS) neurons to change in firing rate in response to tastes and smells. The gustatory (and olfactory) GS neurons, compared to the non-gustatory GS or both types of GIS cells, displayed significantly higher sensitivities to catecholamines. Neurons with both "endogenous" and "exogenous" chemosensitivity were found to be topographically organized in the LHA, AMY and GP as well. While receiving further evidence for the substantial morphological and functional overlapping of the brain's glucose-monitoring neural network and the central gustatory representations, on the basis of the present and previous findings, it is suggested that constituents of this complex system accomplish a simultaneous monitoring, integration and control of a broad variety of feeding-associated signals of the internal and external milieux for the biological welfare of the organism.

Role of forebrain glucose-monitoring neurons in the central control of feeding: I. Behavioral properties and neurotransmitter sensitivities.

Extracellular single neuron recording experiments were performed in the lateral hypothalamic area (LHA), amygdaloid body (AMY) and globus pallidus (GP) of anesthetized rats and anesthetized or alert rhesus monkeys during microelectrophoretic administration of different neurochemicals including glucose. Neuron activity in the behaving primate was also investigated during a conditioned bar press alimentary task, as well as during presentation of food and non-food objects. In the LHA, AMY and GP specific glucose-sensitive (GS) neurons were found, as their activity were suppressed by glucose. The proportion of GS neurons was approximately 29%, 11% and 14%, respectively. The GS neurons in the monkey were especially likely to respond to phase of the conditioned alimentary task, and these same neurons appeared to be particularly influenced by sensorimotor and motivational factors. LHA, AMY and GP GS neurons displayed distinct sensitivities to various neurotransmitters applied microelectrophoretically. The present results, along with previous data, indicate that a hirearchically organized network of the brainstem and forebrain glucose-monitoring neurons exit and this system is involved in the regulation of feeding.

Glucose-sensitive neurons of the globus pallidus: I. Neurochemical characteristics.

The lateral hypothalamic area (LHA) and globus pallidus (GP) are basically involved in the regulation of feeding and metabolic processes. In the LHA, glucose-sensitive (GS) neurons were described: their activity was found to be specifically suppressed by electrophoretic application of glucose, and these neurons appeared to be also influenced by various feeding-associated neurochemical signals. The main goal of the present experiments was to examine whether similar GS neurons exist in the GP. In addition, neurochemical attributes of the cells were also tested. In anesthetized rats and anesthetized or awake monkeys, single-neuron activity of the GP was recorded by means of carbon fiber multibarreled microelectrodes and the effects of glucose, glutamate (Gt), GABA, dopamine (DA), noradrenaline (NA) and acetylcholine (Ach) were studied. In both the rat and monkey GP, approximately 12% of the neurons examined responded, with inhibition, to glucose. GP neurons, in a high proportion, were also inhibited by GABA and NA. After application of Gt, DA, or Ach, activity increase or decrease occurred. GS neurons exhibited remarkable sensitivity to these neurochemicals previously identified as neurotransmitters of the complex pallidal, extrapyramidal-limbic neuron loops. The results, along with previous data, indicate that GS cells of the GP, while possessing complex neurochemical characteristics, may belong to a hierarchically organized central glucose-monitoring system essential in the regulation of feeding.

Glucose-sensitive neurons of the globus pallidus: II. Complex functional attributes.

The globus pallidus (GP) is intimately involved in regulation of various aspects of hunger- and thirst-motivated behaviors. Our parallel neurochemical studies demonstrated the existence of GP neurons whose discharge rates are suppressed by glucose applied microelectrophoretically. In the present series of experiments, we aimed to provide complex, feeding-associated functional characterization--similar to that previously accomplished in the case of lateral hypothalamic and amygdaloid chemosensitive neurons--of these glucose-sensitive (GS) and the glucose-insensitive (GIS) pallidal cells. To do so, extracellular single neuron activity of the GP was recorded in anesthetized rats and anesthetized or awake rhesus monkeys by means of carbon fiber, multibarreled glass microelectrodes during: a) microelectrophoretic administration of chemicals, b) gustatory, and c) olfactory stimulations. In alert primates, activity changes were also recorded during presentation of food and nonfood objects as well as during the performance of a conditioned, high fixed-ratio bar-press feeding task. The half of pallidal cells examined showed firing rate changes during phases of the conditioned alimentary task. In both species, about 1/7 of all neurons tested proved to be GS, while the proportion of cells responding to gustatory and olfactory stimulations was 19% and 16%, respectively. Task-related and taste- and smell-responsive units were mainly found among the GS neurons of the pallidum. These data, along with previous findings, indicate that chemosensitive cells of the GP, in an apparent overlap with units of the central gustatory representation, are involved in a hierarchically organized glucose-monitoring neural network, through which pallidal neurons exert their integrative functions in the central feeding control.

Neuronal damage following transient cerebral ischemia and its restoration by neural transplant.

The middle cerebral artery (mca) was intraluminally occluded for one hour prior to reperfusion in the rat. Neuronal damage as well as motor imbalance were assessed in both acute and chronic stages with or without neural transplant in the striatum. In acute stage, argyrophil III staining demonstrated "collapsed" dark neurons in the ipsilateral striatum, cortex, reticular thalamus, amygdala and sometimes in the hippocampus. They had shrunken somata and corkscrew-like dendrites. In accordance with the appearance of dark neurons, the immunoreactivity for calpain of endogenous inactive form decreased or disappeared in ischemic areas. In chronic stage, ischemic core area (striatum and cortex) got into porencephaly, and animals made rotations following methamphetamine injection. Neural transplant (fetal striatal cells) was made during 2 to 4 weeks after the ischemia. Once the transplant survived and grew in the striatum, the methamphetamine rotations were attenuated. Using mca ischemic model rats we report here pathophysiological processes that lead to neuronal damage and infarct. Neural transplants into these animals brought partial restoration in motor disturbance, offering a valuable information concerning therapeutic possibility.