Blockade of micro-opioid receptors in the medial thalamus inhibits acquisition, but not expression, of morphine-induced conditioned place preference.

The medial thalamus contains abundant mu-opioid receptors and is activated by acute morphine administration. However, the role of the medial thalamus in the rewarding effects of morphine is unclear. The present study examined whether mu-opioid receptors of the medial thalamus influenced the acquisition and expression of morphine-induced conditioned place preference (CPP) in rats. An unbiased apparatus and biased subject assignment were used. Administration of morphine in increasing doses (2 mg/kg, 4 mg/kg, 6 mg/kg, 10 mg/kg, s.c.) was paired with an initially non-preferred chamber and saline administration was paired with an initially preferred chamber. Conditioning trials were conducted twice daily for 4 days. Microinjection of the irreversible mu-opioid receptor antagonist, beta-funaltrexamine (5 microg/rat), into the medial thalamus 23 h prior to each morphine conditioning completely blocked the acquisition of CPP. However, microinjection of beta-funaltrexamine into the medial thalamus after morphine conditioning trials, but 23 h prior to a test session, had no effect on the expression of CPP. It is concluded that mu-opioid receptors in the rat medial thalamus are involved in the acquisition, but not expression, of morphine-induced CPP.

Fos and glutamate AMPA receptor subunit coexpression associated with cue-elicited cocaine-seeking behavior in abstinent rats.

Cocaine-associated cues acquire incentive motivational effects that manifest as craving in humans and cocaine-seeking behavior in rats. We have reported an increase in neuronal activation in rats, measured by Fos protein expression, in various limbic and cortical regions following exposure to cocaine-associated cues. This study examined whether the conditioned neuronal activation involves glutamate AMPA receptors by measuring coexpression of Fos and AMPA glutamate receptor subunits (GluR1, GluR2/3, or GluR4). Rats trained to self-administer cocaine subsequently underwent 22 days of abstinence, during which they were exposed daily to either the self-administration environment with presentations of the light/tone cues previously paired with cocaine infusions (Extinction group) or an alternate environment (No Extinction group). All rats were then tested for cocaine-seeking behavior (i.e. responses without cocaine reinforcement) and Fos and AMPA glutamate receptor subunits were measured postmortem using immunocytochemistry. The No Extinction group exhibited increases in cocaine-seeking behavior and Fos expression in limbic and cortical regions relative to the Extinction group. A large number of Fos immunoreactive cells coexpressed GluR1, GluR2/3, and GluR4, suggesting that an action of glutamate at AMPA receptors may in part drive cue-elicited Fos expression. Importantly, there was an increase in the percentage of cells colabeled with Fos and GluR1 in the anterior cingulate and nucleus accumbens shell and cells colabeled with Fos and GluR4 in the infralimbic cortex, suggesting that within these regions, a greater, and perhaps even different, population of AMPA receptor subunit-expressing neurons is activated in rats engaged in cocaine-seeking behavior.

Morphine induction of c-fos expression in the rat forebrain through glutamatergic mechanisms: role of non-n-methyl-D-aspartate receptors.

Acute injection of morphine induces expression of the immediate-early genes c-Fos and JunB in several forebrain regions of the rat, in part through an N-methyl-D-aspartate (NMDA) receptor-dependent mechanism. Because membrane depolarization through (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors is believed to be necessary for full activation of NMDA receptors, we determined the role of AMPA receptors in morphine-induced c-Fos expression. Rats were given the AMPA receptor antagonist GYKI-52466 (12.9 mg/kg, i.p.) 15 min before morphine (10 mg/kg, s.c.), or the AMPA receptor enhancer CX516 (30 mg/kg, i.p.) 5 min after morphine. The c-Fos response was attenuated by the antagonist and augmented by the enhancer. Using double immunocytochemistry, we found that morphine induced c-Fos in neurons containing the GluR2/3, but not the GluR1 and rarely the GluR4, subunits of the AMPA receptor. Double immunocytochemistry for mu opioid receptor and c-Fos showed that c-Fos expression was mainly absent in the patch compartment of the striatum, which is enriched in mu opioid receptors. The glutamatergic synapse often contains metabotropic receptors as well as ionotropic receptors. Type I metabotropic glutamate receptors are coupled to activation of protein kinase C, which has also been shown to mediate the immediate-early gene response to morphine. To determine if activation of metabotropic glutamate receptors is involved in rapid effects of morphine on the brain, rats were given the type I metabotropic glutamate receptor antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 0.2 mg/kg, i.p.) or vehicle 30 min before morphine treatment. Pretreatment with AIDA completely blocked morphine-induced c-Fos expression in the caudate-putamen.Taken together, these results demonstrate involvement of both AMPA and type I metabotropic glutamate receptors in the acute effects of morphine on the forebrain, supporting an important role for glutamatergic neurotransmission mediated by non-NMDA glutamate receptors in morphine's actions.

Involvement of nitric oxide in morphine-induced c-Fos expression in the rat striatum.

Induction of expression of immediate-early gene c-Fos in the striatum is a common effect of many drugs of abuse, including morphine. Previous studies have shown that the morphine-mediated c-Fos response is attenuated by antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. Other evidence suggests that the NDMA receptor may be coupled to the enzyme neuronal nitric oxide synthase (nNOS). NMDA receptor-mediated increases in intracellular calcium can activate nNOS, which catalyzes the formation of the signaling molecule nitric oxide. Because activation of NMDA receptors mediates morphine-induced c-Fos expression, we tested the hypothesis that activation of nNOS is involved in this cascade. Male rats were injected with the nNOS-selective inhibitor 7-nitroindazole (7-NI) or vehicle 30 min prior to injection of morphine sulfate or vehicle. Two hours later they were perfused with fixative and the brains removed for immunocytochemical analysis for c-Fos. Morphine induced c-Fos expression in the striatum, cerebral cortex, and midline/intralaminar nuclei of thalamus. Expression in the striatum, but not thalamus or cortex, was significantly blocked by 7-NI. Double-label immunocytochemistry revealed no co-localization of c-Fos and nNOS in any brain region. These results support a role for nNOS in the neural circuits activated by morphine.

Deafferentation-induced changes in protein kinase C expression in the rat cochlear nucleus.

Isoforms of the signal transducing molecule, protein kinase C (PKC), may play a role in neural plasticity following sensory deafferentation. To explore the role of PKC in central auditory plasticity, we studied the effect of auditory deafferentation on the expression of PKC betaI, betaII, gamma, and delta in the rat dorsal (DCN) and ventral cochlear nucleus (VCN), using immunocytochemistry. Male rats were treated with kanamycin and furosemide to induce hair cell loss. At various intervals post-treatment, brains were perfusion-fixed and processed for immunocytochemistry. Following deafferentation, we observed a gradual increase in PKC betaI immunoreactivity (ir) in the deepest layers of the DCN, possibly representing synapses of primary afferents or parallel fibers on unlabeled neurons. Correlated with this, we observed an increase in the number of neurons in the deep DCN that showed PKC delta ir. In controls, we observed PKC gamma ir in small ovoid cells concentrated in the middle layer of the DCN. From days 4 through 14 after deafferentation, we found an increase in the intensity of staining of these cells, with a return toward control levels by day 28. Finally, Purkinje-like cells (PLC) in the VCN, which express only PKC delta in control rats, began to express PKC gamma after deafferentation, correlated with increased expression of calbindin D28k in PLC. Thus PKC isoforms are differentially regulated in the CN following deafferentation, supporting a role for PKC in auditory plasticity.

Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy.

We developed a method of double immunocytochemistry (ICC) that can be used with conventional light microscopy for localizing two different nuclear proteins. The procedure involves two sequential rounds of ICC that both employ the avidin and biotin conjugated enzyme (ABC) amplification method, separated by an Avidin D and biotin blocking step to reduce non-specific avidin-biotin reactions. Round one of ICC employs the use of avidin and biotin conjugated alkaline phosphatase (ABC-AP) and the Vector Red (VR) substrate, which produces a red colorimetric reaction product. The second round of ICC makes use of avidin and biotin conjugated peroxidase (ABC-HRP) and the Vector(R) SG substrate, which produces a gray colorimetric reaction product. Neuronal nuclei that are double-labeled for both proteins appear red with a gray core. This protocol allows the simultaneous detection of two proteins within the same subcellular compartment of a single neuron, without the need for epifluorescence or scanning confocal laser microscopy.

Androgenic-anabolic steroids blunt morphine-induced c-fos expression in the rat striatum: possible role of beta-endorphin.

Self-administration of large doses of androgenic-anabolic steroids (AAS) in a significant portion of the population suggests that these agents are drugs of abuse. However, acute administration of AAS did not induce striatal immediate-early genes (IEG) expression in male rats, indicating that AAS do not share a common mechanism of action with other drugs of abuse. Surveys have indicated that people who abuse AAS are more likely to self-administer other drugs of abuse than do people who do not take AAS. In the present study, chronic administration of AAS blunted the striatal c-fos response to morphine, indicating that AAS can alter the molecular responses to at least one drug of abuse. Chronic administration of AAS also increased the content of beta-endorphin in the midline thalamus, suggesting a possible mechanism by which AAS may modulate the response to morphine through regulation of thalamo-striatal neurons.

Role of the dorsal raphe nucleus in morphine-induced immediate early gene expression in the rat striatum.

Serotonin (5-HT) is thought to be involved in morphine action in the brain. To determine if the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN) are involved in morphine-induced c-Fos and JunB expression in the caudate-putamen (CPu), the mu receptor antagonist, beta-funaltrexamine (beta-FNA), was unilaterally infused into the PAG adjacent to DRN prior to morphine. Behaviorally, beta-FNA prevented morphine-induced loss of righting and Straub tail. In the CPu of beta-FNA treated rats, morphine-induced c-Fos and JunB were attenuated compared to vehicle-infused rats. These results suggest that morphine acts within the PAG-DRN to exert rapid behavioral effects and to induce c-Fos and JunB in the striatum.

Sexual dimorphism in the response to N-methyl-D-aspartate receptor antagonists and morphine on behavior and c-Fos induction in the rat brain.

It has been suggested that there are sex differences in the neural response to drugs of abuse. Previous studies have shown that, upon administration of morphine, the immediate early gene c-Fos is induced in the striatum, nucleus accumbens and cortex of the rat brain. This induction of c-Fos is reduced by administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate. However, in studies using immunocytochemistry, we found that the pattern of this expression differed markedly between the sexes. In male rats treated with morphine (10 mg/kg, s.c.) and killed 2 h later, there was an induction of c-Fos in the dorsomedial caudate-putamen, the nucleus accumbens and in the intralaminar nuclei of the thalamus. Administration of dizocilpine maleate (0.2 mg/kg, i.p.; 30 min before morphine) partially blocked the response in the caudate-putamen, but not in the thalamus. In females, morphine induced c-Fos in the caudate-putamen, but with more inter-animal variability than in males. In the midline intralaminar thalamic nuclei, female rats showed less induction than males. In male rats, dizocilpine maleate alone caused negligible induction of c-Fos, whereas in female rats, it caused a large induction in the rhomboid, reuniens and central medial nuclei of the thalamus, and in the cortex. Whereas dizocilpine maleate partially blocked the morphine-induced c-Fos expression in the caudate-putamen of males, it completely blocked this response in females. With dizocilpine maleate alone, there was little or no effect on behavior in male rats, whereas in female rats, it caused head bobbing, thrashing, hyperactivity and uncoordinated movements. These behavioral sex differences were not seen on treatment of rats with the competitive N-methyl-D-aspartate receptor antagonist 2R,4R,5S-2-amino-4,5-(1,2-cyclohexyl)-7-phosphoheptanoic acid (NPC-17742; 10 mg/kg, i.p.) and this drug did not induce c-Fos expression in either sex. In the caudate-putamen, morphine-induced c-Fos expression was significantly reduced by NPC-17742 (30 min before morphine) in males and completely blocked in females. These results suggest that the responses to both morphine and N-methyl-D-aspartate receptor antagonists differ between the sexes and emphasize that glutamate is involved in morphine-induced immediate early gene expression in the brain. These studies thus have important implications for gender differences in drug addiction.

Infusion of beta-FNA into the thalamus attenuates morphine-induced c-Fos induction in the rat caudate putamen.

The medial thalamus contains mu opioid receptors and sends a glutamatergic projection to the caudate putamen (CPu) in rat. Morphine-induced c-Fos expression in the CPu has been shown to be blocked by pretreatment with antagonists to N-methyl-D-aspartate receptors, indicating the involvement of glutamate in this morphine-induced response. The importance of the glutamatergic projections from the thalamus was assessed by infusing the mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), prior to systemic morphine injection. Infusion of beta-FNA near specific medial thalamic nuclei attenuated morphine-induced c-Fos expression in the CPu.

Chronic administration of morphine alters immediate-early gene expression in the forebrain of post-dependent rats.

Previous studies from this laboratory have demonstrated that acute, systemic administration of morphine results in an induction of the immediate-early gene (IEG) proteins, c-Fos and Jun-B, in the dorsomedial portion of the rat caudate-putamen (CPu). These studies have also shown that morphine can induce c-Fos in the central medial nucleus of the thalamus (CM). To determine whether this response is altered in post-dependent rats, twice-daily injections of an ascending dose of morphine were administered for 5 days, followed by a withdrawal period of 7 or 14 days. A challenge injection of morphine (10 mg/kg) was administered on the last day of withdrawal. As compared to an acute dose of morphine in a naive animal, the induction of c-Fos was increased in the dorsolateral CPu following challenge injection at 7 days, but not at 14 days. Induction of c-Fos in the CM following the challenge injection was blunted following 7 day, but not at 14 days, of withdrawal. An increase in the IEG protein, Jun-B, was also seen following 7 but not 14 days of withdrawal in both the dorsomedial and dorsolateral CPu. These findings demonstrate that a chronic treatment of morphine can result in altered patterns of IEG expression upon challenge with acute morphine, in a time-dependent manner, within the rat CPu and CM.

Tests used to assess the cognitive abilities of aged rats: their relation to each other and to hippocampal morphology and neurotrophin expression.

BACKGROUND: Aged rodents have proven to be a useful tool in studying age-related cognitive decline, particularly with regard to hippocampal function. A number of maze tests have been developed to evaluate hippocampal function in aged rodents, including the eight-arm radial maze, Barnes circular platform maze and Morris water maze. To some extent, these mazes have been used interchangeably to evaluate aged animals. Few researchers, however, have examined how performance of individual, aged animals compares in these three mazes. OBJECTIVE: The purpose of this study was to compare the performances in the three mazes and to examine how such performances are related to each other, to hippocampal morphology and to neurotrophin gene expression. METHODS: We screened groups of young and old Fisher 344 x Brown Norway rats for general health and physical abilities, tested the animals in the three mazes and examined correlations among performances in the mazes and in screening tests. Hippocampal neuron density and expression of hippocampal neurotrophin mRNAs were also examined and compared with behavior in the three mazes. RESULTS: Aged animals were found to be impaired in all three mazes and to have lower hippocampal neuron densities compared with young animals, with poor learning behavior significantly correlating with reduced hippocampal neuron density. Differences were observed between performance in the different mazes, but in general the Morris water maze and Barnes circular platform maze were found to give similar results.

The 5-HT3 receptor antagonist, MDL 72222, dose-dependently potentiates morphine-induced immediate-early gene expression in the rat caudate putamen.

Previous studies from this laboratory have demonstrated that acute administration of morphine induces the immediate-early genes (IEGs) c-Fos and JunB in the rat caudate putamen (CPu). In the present study, we tested the hypothesis that the serotonin-3 receptor (5-HT3R) is involved in morphine-induced IEG expression, using the selective antagonist to the 5-HT3R, MDL 72222. Rats were divided into three pretreatment groups: MDL 72222, 1 mg/kg or 10 mg/kg; or vehicle (DMSO). Thirty minutes following the pretreatment, the rats were administered either morphine (10 mg/kg) or vehicle. Morphine significantly induced c-Fos expression in the dorsomedial CPu, as we have reported previously. Whereas MDL 72222 alone did not induce c-Fos, it potentiated the morphine-induced c-Fos expression. Morphine also induced JunB expression in the same region of the dorsomedial CPu. At 1 mg/kg, MDL 72222 both induced JunB expression and potentiated the response induced by morphine. At 10 mg/kg, MDL 72222 had no effect on basal JunB levels, but augmented the response to morphine. These findings demonstrate that the 5-HT3R antagonist, MDL 72222, can positively modulate morphine-induced IEG expression in the rat CPu in a dose dependent manner, in contrast to the reported suppressive effect observed when this antagonist is administered prior to amphetamine.

Drugs of abuse and immediate-early genes in the forebrain.

A diverse array of chemical agents have been self administered by humans to alter the psychological state. Such drugs of abuse include both stimulants and depressants of the central nervous system. However, some commonalties must underlie the neurobiological actions of these drugs, since the desire to take the drugs often crosses from one drug to another. Studies have emphasized a role of the ventral striatum, especially the nucleus accumbens, in the actions of all drugs of abuse, although more recent studies have implicated larger regions of the forebrain. Induction of immediate-early genes has been studied extensively as a marker for activation of neurons in the central nervous system. In this review, we survey the literature reporting activation of immediate-early gene expression in the forebrain, in response to administration of drugs of abuse. All drugs of abuse activate immediate-early gene expression in the striatum, although each drug induces a particular neuroanatomical signature of activation. Most drugs of abuse activate immediate-early gene expression in several additional forebrain regions, including portions of the extended amygdala, cerebral cortex, lateral septum, and midline/intralaminar thalamic nuclei, although regional variations are found depending on the particular drug administered. Common neuropharmacological mechanisms responsible for activation of immediate-early gene expression in the forebrain involve dopaminergic and glutamatergic systems. Speculations on the biological significance and clinical relevance of immediate-early gene expression in response to drugs of abuse are presented.

A two focal plane method for digital quantification of nuclear immunoreactivity in large brain areas using NIH-image software.

In principle, digital acquisition of cell-count data from serially-sectioned immunocytochemical material is a straightforward enterprise. First, a serial brain section is magnified by use of a microscope interfaced to a computer. Then, using appropriate hardware and software, a digital image is captured, and cellular profiles of interest are segmented from background objects according to mean grayscale intensity and pixel area. Ideally, the cells of interest would be uniformly distinguishable from other objects or areas of the image, with respect to grayscale intensity and size. However, due to non-uniformity in background staining of neuropil, immunocytochemical material often departs markedly from this ideal situation. As a consequence, determining grayscale intensity and cell size cutoff values which separate cells of interest from background becomes laborious and arbitrary. This problem can be diminished by increasing the magnification of the digitized image, which increases the figure-ground resolution of the image. However, high-magnification images make tissue navigation difficult and require that multiple images be captured. This paper describes a two focal plane procedure for obtaining cell counts from nuclear-stained immunocytochemistry material. This procedure allows the capturing and cell counting of relatively low-magnification images with high digital figure-ground resolution.

Tumor necrosis factor-alpha: a neuromodulator in the CNS.

In the central nervous system (CNS), the cytokine tumor necrosis factor-alpha (TNF alpha) is produced by both neurons and glial cells, participates in developmental modeling, and is involved in many pathophysiological conditions. There are activity-dependent expressions of TNF alpha as well as low levels of secretion in the resting state. In contrast to the conventional view of a cytotoxic effect of TNF alpha, accumulating evidence suggests a beneficial effect when TNF alpha is applied at optimal doses and at specific periods of time. The bimodal effect is related to subtypes of receptors, activation of different signal transduction pathways, and the presence of other molecules that alter the intracellular response elements such as immediate-early genes. TNF alpha may be an important neuromodulator in development of the CNS, diseases of demyelination and degeneration, and in the process of regeneration. It could induce growth-promoting cytokines and neurotrophins, or it could increase the production of antiproliferative cytokines, nitric oxide, and free radicals, thereby contributing to apoptosis.

Aging in the hippocampus: interrelated actions of neurotrophins and glucocorticoids.

Over the past two decades, evidence has been accumulating that diffusible molecules, such as growth factors and steroids hormones, play an important part in neural senescence, particularly in the hippocampus. There is also evidence that these molecules do not act as independent signals, but show interrelated regulation and cooperative control over the aging process. Here, we review some of the changes that occur in the hippocampus with age, and the influence of two classes of signaling substances: glucocorticoids and neurotrophins. We also examine the interactions between these substances and how this could influence the aging process.

Protein kinase C in central auditory pathways of the rat.

Protein kinase C is an important intracellular signaling molecule. Many of its ten isoforms are highly expressed in brain, and protein kinase C has been implicated in the regulation of the activity of receptors of several major neurotransmitters, including glutamate, acetylcholine, glycine, and gamma-aminobutyric acid. These neurotransmitters and their receptors are present in central auditory pathways, suggesting their role in auditory signal processing. Although they may be important modulators of the function of these neurotransmitter receptors, the distribution of protein kinase C isoforms in central auditory systems has not been well characterized. By using immunocytochemistry with specific antibodies, we studied the distribution of immunoreactivity of four isoforms of protein kinase C, betaI, betaII, gamma, and gamma, in central auditory systems of rat brain. Each of these protein kinase C isoforms was found to have a unique distribution in the auditory brainstem and cortex, supporting a role for these isoforms of protein kinase C in different aspects of auditory sensory processing.

Protein kinase C in central vestibular, cerebellar, and precerebellar pathways of the rat.

The protein kinase C family of enzymes is composed of at least ten different isoforms that display a variety of distinct biochemical specificities. Many of these isoforms are highly expressed in brain, and some show regional specificity in their distribution, suggesting that they may serve specific functions. By using immunocytochemistry to localize the betaI, betaII, gamma, or delta isoforms of protein kinase C in the central vestibular system of the adult rat, we found the vestibular ganglion and its peripheral and central processes of the eighth nerve to be heavily labeled with protein kinase C betaI immunoreactivity. Labeled axons and terminals were also found in all four vestibular nuclei. Some neurons of the vestibular ganglion were weakly stained with the antibody to protein kinase C betaII, as were scattered axons in the eighth nerve, and scattered axons and terminals were found in all four vestibular nuclei among weakly labeled neurons. A few axons in the vestibular portion of the eighth nerve were labeled with protein kinase C gamma immunoreactivity, and neurons of the spinal, lateral, and superior vestibular nuclei were heavily decorated with synapses, presumably derived from Purkinje neurons, which were also strongly immunoreactive. Neurons of the medial vestibular nucleus were not as heavily innervated. With the antibody to protein kinase C delta, we found scattered, weakly immunoreactive neurons in the vestibular portion of the eighth nerve. Myelinated fiber bundles of the spinal vestibular nucleus contained moderate numbers of labeled axons, and the other vestibular nuclei were well innervated by protein kinase C delta axons and terminals. Most of these probably derive from Purkinje cells, which were labeled in longitudinal bands interspersed with bands of labeled basket cells. These data suggest that particular protein kinase C isoforms play specific roles in vestibular and cerebellar function.