Meredith M. Garcia

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.

Alterations in immediate-early gene proteins in the rat forebrain induced by acute morphine injection.

Injection of morphine (10 mg/kg) induced a complex immediate-early gene response in the rat forebrain, as detected with immunocytochemistry. The c-Fos protein was induced consistently in the dorsomedial caudate-putamen, the nucleus accumbens, and in midline and intralaminar nuclei of the thalamus. In some rats induction was also seen in the parietal and insular cortex and in lateral regions of the caudate-putamen. Induction was detectable, although weak, at 30 min, was maximal at 2 h, and was undetectable 3 h after injection. JunB was induced in the same regions of the caudate-putamen as found for c-Fos, but was not induced in the nucleus accumbens or thalamus. In the caudate-putamen, JunB induction was still present 3 h after injection. A considerably smaller induction of c-Jun was noted in the dorsomedial caudate-putamen and in deep neocortex. Expression of JunD was inhibited in intralaminar and midline thalamic nuclei. Increases in numbers of cells immunoreactive for a Jun-related antigen (Jra) were found in the caudate-putamen and nucleus accumbens. These results indicate a complex immediate-early gene response to acute morphine, suggesting that morphine activates or inhibits specific neurons and circuits in the forebrain.

The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells ( approximately 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the alpha9-nicotinic response, suggesting an enzymatic interaction with the alpha9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha9nAChR, SK, or both.

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.

Glucocorticoid regulation of preproenkephalin gene expression in the rat forebrain.

The effects of glucocorticoids on the levels of preproenkephalin (PPE) mRNA in the rat forebrain were analyzed with in situ hybridization and dot blots. In adrenally-intact rats, high levels of PPE mRNA, as assessed by in situ hybridization, were localized in the caudate-putamen, nucleus accumbens, central amygdala, and ventrolateral ventromedial hypothalamus (VMHVL), and low levels in the hippocampus. After adrenalectomy, the density of PPE mRNA-positive cells and the level of PPE mRNA/cell were decreased in all regions except the hippocampus. Acute treatment with corticosterone (CORT) in adrenalectomized rats increased the level of PPE mRNA/cell in the caudate-putamen and VMHVL. In intact rats, chronic treatment with CORT increased the density of PPE mRNA-positive cells in the caudate-putamen and hippocampus, and the level of PPE mRNA/cell in the caudate-putamen and nucleus accumbens. The effect of chronic CORT treatment on PPE mRNA in the striatum, amygdala, hippocampus and mediobasal hypothalamus was assessed with dot blots. Chronic CORT treatment increased PPE mRNA levels in the caudate-putamen and hippocampus. There was a good correlation between results on the effect of chronic CORT treatment on PPE mRNA levels in intact rats, obtained from dot blots and in situ hybridization. Results from this study suggest that glucocorticoids are required for the maintenance of basal PPE mRNA levels in most regions of the rat forebrain. There is, however, considerable regional heterogeneity in the effect of glucocorticoid treatment on PPE mRNA levels in adrenalectomized and intact rats. Increased PPE mRNA levels in response to high circulating levels of glucocorticoids, e.g. in stress, may have important pathophysiological consequences.

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.

The metabotropic glutamate receptors of the vestibular organs

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.

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.

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.

Charting of Jun family member proteins in the rat forebrain and midbrain: immunocytochemical evidence for a new Jun-related antigen

Immunocytochemistry was used to localize members of the Jun family of immediate-early genes in the forebrain and midbrain of non-stimulated male rats. Antibodies against specific peptide sequences of c-Jun (Ab-1 and Ab-2 from Oncogene Science) and against expressed proteins of JunB and JunD (both from Dr. R. Bravo) revealed widespread and unique distributions for each of these antigens. Charts were made of the distribution of each antigen, and extensive comparisons were made of previous results obtained using in situ hybridization to localize mRNAs for c-jun, junB and junD. Our results indicate a generally favorable comparison between immunoreactivity and distribution of mRNAs for JunB and JunD, but in the case of c-Jun, immunoreactivity and mRNA were comparable only with the Ab-1 antibody. Indeed, the immunocytochemical distribution of the antigen recognized by the c-Jun Ab-2 antibody was distinctly different from that of the other Jun proteins or mRNAs in the rat brain. This antibody (Ab-2) recognized a nuclear protein found extensively in the caudate-putamen, nucleus accumbens, layer II of the olfactory tubercle, the central nucleus of the amygdala, and the lateral division of the bed nucleus of the stria terminalis. Scattered labeled nuclei were found in a few other forebrain structures. Within the caudate-putamen, immunoreactivity was restricted to the matrix compartment, as determined by immunostaining of adjacent sections with the matrix-marker calbindin D28k. Western blots of caudate-putamen demonstrated that this antibody recognized a protein doublet of molecular masses approximately 37 and 34 kDa, distinct from the molecular masses of c-Jun, JunB and JunD. This unique neuroanatomical distribution and molecular mass suggests that this antibody recognizes a previously undescribed Jun-related antigen.