Huda Akil

Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data

Genome-wide expression profiling is a powerful tool for implicating novel gene ensembles in cellular mechanisms of health and disease. The most popular platform for genome-wide expression profiling is the Affymetrix GeneChip. However, its selection of probes relied on earlier genome and transcriptome annotation which is significantly different from current knowledge. The resultant informatics problems have a profound impact on analysis and interpretation the data. Here, we address these critical issues and offer a solution. We identified several classes of problems at the individual probe level in the existing annotation, under the assumption that current genome and transcriptome databases are more accurate than those used for GeneChip design. We then reorganized probes on more than a dozen popular GeneChips into gene-, transcript- and exon-specific probe sets in light of up-to-date genome, cDNA/EST clustering and single nucleotide polymorphism information. Comparing analysis results between the original and the redefined probe sets reveals ∼30–50% discrepancy in the genes previously identified as differentially expressed, regardless of analysis method. Our results demonstrate that the original Affymetrix probe set definitions are inaccurate, and many conclusions derived from past GeneChip analyses may be significantly flawed. It will be beneficial to re-analyze existing GeneChip data with updated probe set definitions.

Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications

The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization.

Anatomy of CNS opioid receptors

Abstract There is a wide body of evidence to suggest the existence of at least three distinct opioid receptor types in the CNS, referred to as μ, δ, and κ. This paper reviews some of the findings that have led to this conclusion and the anatomical distributions of these sites in the rat brain. Their relation to the opioid peptides and some of the proposed functions mediated by these receptor systems are also discussed.

Pattern and time course of immediate early gene expression in rat brain following acute stress

The pattern and time course of brain activation in response to acute swim and restraint stress were examined in the rat by in situ hybridization using complementary RNA probes specific for transcripts encoding the products of the immediate early genes c-fos, c-jun and zif/268. A widespread pattern of c-fos messenger RNA expression was detected in response to these stressors; surprisingly, the expression patterns were substantially similar following both swim and restraint stress. A dramatic induction of c-fos messenger RNA was observed in numerous neo- and allocortical regions, the lateral septal nucleus, the hypothalamic paraventricular and dorsomedial nuclei, the anterior hypothalamic area, the lateral portion of the retrochiasmatic area, the medial and cortical amygdaloid nuclei, the periaqueductal gray, and the locus coeruleus; however, a prominent induction of c-fos was also seen in numerous additional subcortical and brainstem regions. Although not as widely expressed in response to stress as c-fos, induction of zif/268 messenger RNA was also detected throughout many brain areas; these regions were largely similar to those in which c-fos was induced, although in a number of regions zif/268 was expressed in regions devoid of c-fos messenger RNA. Few brain areas showed increased expression of c-jun following stress; these regions also showed induction of c-fos and/or zif/268. The time courses of expression of all three immediate early genes were similar, with peak levels observed at the 30 or 60 min time point, and a markedly reduced signal evident at 120 min post-stress. However, in a number of cases a delayed and/or prolonged induction was noted that may be indicative of secondary neuronal activation. A number of recent studies have attempted to define neural pathways which convey stress-related information to the hypothalamic-pituitary-adrenal axis. The present results reveal a widespread pattern of neuronal activation in response to acute swim or restraint stress. These findings may aid in the identification of stress-specific neural circuits and are thus likely to have important implications for our understanding of neuronal regulation of the stress response.

A selective role for dopamine in stimulus-reward learning

Individuals make choices and prioritize goals using complex processes that assign value to rewards and associated stimuli. During Pavlovian learning, previously neutral stimuli that predict rewards can acquire motivational properties, becoming attractive and desirable incentive stimuli. However, whether a cue acts solely as a predictor of reward, or also serves as an incentive stimulus, differs between individuals. Thus, individuals vary in the degree to which cues bias choice and potentially promote maladaptive behaviour. Here we use rats that differ in the incentive motivational properties they attribute to food cues to probe the role of the neurotransmitter dopamine in stimulus–reward learning. We show that intact dopamine transmission is not required for all forms of learning in which reward cues become effective predictors. Rather, dopamine acts selectively in a form of stimulus–reward learning in which incentive salience is assigned to reward cues. In individuals with a propensity for this form of learning, reward cues come to powerfully motivate and control behaviour. This work provides insight into the neurobiology of a form of stimulus–reward learning that confers increased susceptibility to disorders of impulse control.

Cloning and pharmacological characterization of a rat μ opioid receptor

We have isolated a rat cDNA clone that displays 75% amino acid homology with the mouse delta and rat kappa opioid receptors. The cDNA (designated pRMuR-12) encodes a protein of 398 amino acids comprising, in part, seven hydrophobic domains similar to those described for other G protein-linked receptors. Data from binding assays conducted with COS-1 cells transiently transfected with a CMV mammalian expression vector containing the full coding region of pRMuR-12 demonstrated mu receptor selectivity. In situ hybridization mRNA analysis revealed an mRNA distribution in rat brain that corresponds well to the distribution of binding sites labeled with mu-selective ligands. Based upon these observations, we conclude that pRMuR-12 encodes a mu opioid receptor.

Altered cortical glutamatergic and GABAergic signal transmission with glial involvement in depression

Abnormalities in l-glutamic acid (glutamate) and GABA signal transmission have been postulated to play a role in depression, but little is known about the underlying molecular determinants and neural mechanisms. Microarray analysis of specific areas of cerebral cortex from individuals who had suffered from major depressive disorder demonstrated significant down-regulation of SLC1A2 and SLC1A3, two key members of the glutamate/neutral amino acid transporter protein family, SLC1. Similarly, expression of l-glutamate-ammonia ligase, the enzyme that converts glutamate to nontoxic glutamine was significantly decreased. Together, these changes could elevate levels of extracellular glutamate considerably, which is potentially neurotoxic and can affect the efficiency of glutamate signaling. The astroglial distribution of the two glutamate transporters and l-glutamate-ammonia ligase strongly links glia to the pathophysiology of depression and challenges the conventional notion that depression is solely a neuronal disorder. The same cortical areas displayed concomitant up-regulation of several glutamate and GABAA receptor subunits, of which GABAAα1 and GABAAβ3 showed selectivity for individuals who had died by suicide, indicating their potential utility as biomarkers of suicidality. These findings point to previously undiscovered molecular underpinnings of the pathophysiology of major depression and offer potentially new pharmacological targets for treating depression.

Immunohistochemical localization of the cloned μ opioid receptor in the rat CNS

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined mu, delta and kappa 1 receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned mu receptor (335-398) to examine the distribution of the mu receptor-like protein with immunohistochemical techniques. mu receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known mu receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.

Monoaminergic mechanisms of stimulation-produced analgesia

The roles played by the cerebral monoamines (dopamine, noradrenaline and serotonin) in stimulation-produced analgesia (SPA) have been investigated in the rat employing the tail flick test. SPA was elicited through bipolar electrodes chronically implanted in the mesencephalic periaqeductal gray matter, an area previously shown to yield potent and reliable analgesic effects. Four approaches were used to alter transmission in monoamine pathways. (1) Depletion of monoamines by administration of tetrabenazine (TBZ), p-chlorophenylalanine (PCPA), alpha-methyl-para-tyrosine (AMPT), or disulfiram. (2) Replacement of depleted monoamine stores by appropiate precursors (5-HTP or L-DOPA) in combination with a peripheral decarboxylase inhibitor. (3) Potentiation of monoamine systems by administration of precursors to previously untreated animals or by administration of a dopamine receptor stimulator, apomorphine. (4) Blockade of catecholamine receptors by haloperidol or of dopamine receptors by pimozide. These four approaches yielded internally consistent results. Depletion of all 3 monoamines (TBZ) led to a powerful inhibition of SPA. Original levels of SPA were restored by injection of either 5-HTP or L-DOPA. Specific depletion of serotonin (PCPA) caused a reduction in SPA, whereas elevation of serotonin levels (5-HTP) caused an increase in SPA. Dopamine receptor blockade (pimozide) decreased SPA, whereas the precursor (L-DOPA) and a dopamine receptor stimulator (apomorphine) increased SPA. On the other hand, selective depletion of noradrenaline (disulfiram) caused an increase in SPA; and at a time when noradrenaline levels are depressed and dopamine levels are elevated (AMPT + L-DOPA), SPA was seen to be particularly enhanced. thus, dopamine and serotonin appear to facilitate SPA, whereas noradrenaline appears to inhibit it. When a general catecholamine receptor blocker (haloperidol) was employed, SPA was diminished, suggesting that the influence of dopamine in SPA is greater than that of noradrenaline. Most of the drugs used in this study significantly altered SPA at doses which left baseline tail flick latency unaffected. It would appear, therefore, that SPA has a neural substrate at least partly independent of that underlying baseline pain responsiveness. Consideration is given to various ascending and descending monoamine system as possible component paths in this neural substrate of SPA. Finally, the present results are discussed in relation to studies by others on the site and mechanism of morphines analgesic action. Some striking parallels between SPA and morphine analgesia are noted. These suggest the existence of a common pain-inhibitory system in the brain activated by morphine and by focal electrical stimulation.

Dynorphin immunocytochemistry in the rat central nervous system.

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300-400 micrograms) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1-13, 7-17, or 1-17). For comparison, antisera to [Leu]enkephalin (residues 1-5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sites, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.