Research Findings - Basic Research

MDMA ("Ecstasy") Inhibits Rats' Response to Stress

MDMA is known to damage forebrain serotonin neurons. Since the integrity of the central serotonergic system is important for some neurochemical responses to acute stress, neurochemical responses during immobilization stress were measured in male rats that had been administered a neurotoxic dosing regimen of MDMA 7 days earlier. In vivo microdialysis was used to assess extracellular dopamine and serotonin in the dorsal hippocampus and prefrontal cortex during 1 hour of immobilization stress. In saline- treated control rats, serotonin in the hippocampus and serotonin and dopamine in the prefrontal cortex were increased during immobilization stress. Rats pretreated with MDMA, however, showed blunted neurotransmitter responses in the hippocampus and the prefrontal cortex. In the MDMA pretreated rats, basal serotonin levels in the hippocampus, but not the prefrontal cortex, were lower compared to saline-pretreated controls. Stress-induced increases in plasma corticosterone and body temperature were not affected by the pretreatment condition. These findings suggest that depletion of serotonin stores in terminal regions with MDMA compromises the ability of the serotonergic neurons to activate central systems that respond to stressful stimuli. This altered responsiveness may have implications for long-term functional consequences of MDMA abuse as well as the interactions between the serotonergic system and stress. Matuszewich, L., Filon, M.E., Finn, D.A. and Yamamoto, B.K. Altered Forebrain Neurotransmitter Responses to Immobilization Stress Following 3,4-methylenedioxymeth-amphetamine. Neuroscience, 110, pp. 41-48, 2002.

Superoxide Free Radical May Play Role in PCP Effects and in Schizophrenia

Kenneth M. Johnson's group previously showed that repetitive administration of PCP to rats in the perinatal period results in cortical apoptosis (cell death) and a long-lasting deficit in sensorimotor gating. Because these changes are blocked by olanzapine, an atypical antipsychotic agent, the researchers suggested that the effects of perinatal PCP could be used to model certain aspects of schizophrenia. Studies of PCP and NMDA-induced cell death suggested that superoxide could play a role in the pathway leading to cell death after PCP administration. The purpose of the current study was to determine whether the in vivo administration of M40403, a superoxide dismutase mimetic agent (which destroys superoxide), could prevent PCP-induced cortical apoptosis and/or deficits in prepulse inhibition (PPI). Deficits in PPI indicate deficits in sensorimotor gating, and decreased PPI is a clinical sign of schizophrenia. Perinatal rat pups were administered 10 mg/kg PCP on postnatal (PN) days 7, 9, and 11, with or without treatment with 10 mg/kg M40403. Pups were either killed on PN 12 for analysis of various apoptotic markers, or they were assessed for PPI on PN 24 to 26. Treatment with M40403 2 and 24 hours after each PCP treatment prevented PCP-induced increases in indicators of apoptosis in the dorsolateral frontal cortex and in the olfactory cortex. PCP-induced proapoptotic changes in Bax and Bcl-XL (regulators of apoptosis) were also prevented by M40403 treatment. This regimen did not prevent the deficit in PPI caused by PCP treatment, but when the treatment regimen was extended through PN 23, M40403 completely prevented the PCP-induced deficit in PPI. These data suggest that perinatal PCP treatment leads to long-lasting changes in the pathway(s) leading to cell death and behavioral deficits, and that the superoxide radical plays a critical role in the underlying mechanism. Wang, C., McInnis, J., West, J.B., Bao, J., Anastasio, N., Guidry, J., Ye, Y., Salvemini, D., and Johnson, K.M. Blockade of Phencyclidine-induced Cortical Apoptosis and Deficits in Prepulse Inhibition by M40403, A Superoxide Dismutase Mimetic. J. Pharmacol. Exp. Ther., 305, pp. 266-271, 2003.

Importance of Nonpharmacological Factors in Nicotine Self-administration

This paper reviews the work of the authors' laboratory and focuses on factors that influence nicotine self-administration. Nicotine dose, by itself, is not sufficient to explain the high rates of self-administration that is seen in animals and humans. It is concluded that although the direct pharmacological effects of nicotine are important, other factors such as environmental stimuli and conditioned reinforcement also influence rates of self-administration. The authors suggest that nicotine's principle function is to magnify the salience of other reinforcers. The interaction between therapeutic strategies targeting the direct effects of nicotine and those of the associated stimuli and contexts should be studied. Caggiula, A.R., Donny, E.C., Chaudhri, N., Perkins, K.A., Evans-Martin, F.F., and Sved, A.F. Importance of Nonpharmacological Factors in Nicotine Self-administration. Physiology & Behavior, 77, pp. 683-687, 2003.

Rat Strain Differences in Nicotine Self-administration Using an Unlimited Access Paradigm

A 23 hour unlimited access paradigm was used in which several strains of rat (including inbred Lewis rats, inbred Fisher 344 rats, and outbred Holzman rats) lever pressed for nicotine. The ability for each strain to learn and maintain operant nicotine self-administration (0.03 and 0.0075 mg/kg, iv) at several FR schedules (FR1 to FR5) was measured. Rat strains differed both in sensitivity to nicotine dose and ability to maintain different schedules of reinforcement. This work suggests that the Lewis rats are genetically more susceptible to nicotine addiction. Brower, V.G., Fu, Y., Matta, S.G., and Sharp, B.M. Rat Strain Differences in Nicotine Self-administration Using an Unlimited Access Paradigm. Brain Research, 930, pp. 12-20, 2002.

Delta Receptor Antagonism

Past efforts in the design of peptide ligands active at opioid receptors have recognized the utility of introducing structural constraints which will limit the available number of conformations which the peptide can adopt, based on single bond rotations about the bonds beta and gamma (C-2 and C-3) to the alpha carbon (C-1) of a particular amino acid. In this respect, modification of the dipeptide Tyr-Tic (tyrosine-tetrahydroisoquinoline) has been of interest, since this sequence is a minimum length peptide which still retains opioid receptor binding. It has been previously determined that methylating the beta carbon and dimethylating the aromatic ring of tyrosine produced four isomers of beta-methyl-2'-6'-dimethyl Tyr-L-Tic, of which only the 2S,3R isomer showed potent binding at the delta receptor (IC50 of 9 nM), and a very high selectivity ratio relative to the mu receptor. The low energy conformation of this molecule is believed to position the substituted phenolic group trans to the C(alpha)-amino bond. In a recombinant in-vitro cell system (Chinese hamster ovary) overexpressing the human delta receptor, this dipeptide behaved as an inverse agonist, as shown by a concentration dependent reduction in GTPgammaS35 binding. In this functional assay, agonists increased the binding of labeled GTP above baseline activity, and an antagonist produced only baseline activity with increasing concentration. In order to test the dipeptide further, the same GTPgammaS35 assay was performed using mouse brain membrane, where the level of delta receptor is likely lower than in the CHO cell expression system. In the membrane, the response was one of neutral antagonism rather than inverse agonism, i.e., the peptide did not alter the baseline activity of GTP binding by the receptor. Additionally, the dipeptide displayed antagonism of deltorphin II (delta receptor) antinociception, but not DAMGO (mu receptor) antinociception, when pre-administered in-vivo by icv injection, using the mouse tail-flick assay. When administered by itself, the dipeptide did not produce a nociceptive response, in either the tail-flick or radiant heat paw-withdrawal assays. Ligands of the type described above, showing selective in-vivo inhibition of the delta opioid receptor, have significance both as pharmacological tools, as well as leads for the development of agents which can prevent the side effects of mu analgesics. Hosohata, K., Varga, E.V., Alfaro-Lopez, J., Tang, X., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., and Yamamura, H.I. (2S,3R)_-Methyl2'6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] Is a Potent, Selective _-Opioid Receptor Antagonist in Mouse Brain. Journal of Pharmacology and Experimental Therapeutics, 304, pp. 683-688, 2003.

Transcellular Transport of a Highly Polar 3+ Net Charge Opioid Tetrapeptide

Oligopeptides are generally thought to have poor permeability across biological membranes. Recent studies, however, suggest significant distribution of [Dmt1]DALDA (Dmt-D-Arg-Phe-Lys-NH2; Dmt is 2,6-dimethyltyrosine), a 3+ net charge opioid peptide, to the brain and spinal cord after subcutaneous administration. Peptide transporters (PEPT1 and PEPT2) play a major role in the uptake of di- and tripeptides across cell membranes, but their ability to transport tetrapeptides is not clear. The purpose of this study was to determine whether [Dmt1]DALDA can translocate across Caco-2 cell monolayers and whether PEPT1 plays a role in the uptake process. Dr. Szeto and her colleagues showed that [3H][Dmt1]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24-10-5 cm/s. When incubated with Caco-2 cells, [3H][Dmt1]DALDA was detected in cell lysates by 5 min. The internalization of [Dmt1]DALDA was confirmed visually with a fluorescent [Dmt1]DALDA analog (H-Dmt-D-Arg-Phe-dns Dap-NH2; dnsDap is dansyl-L-diaminopropionic acid). The uptake of [3H][Dmt1]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [14C]glycine-sarcosine uptake but increased [3H][Dmt1]DALDA uptake 34-fold. These findings suggest that PEPT1 is not involved in [Dmt1]DALDA internalization. [Dmt1]DALDA uptake was also observed in SH-SY5Y, human embryonic kidney 293, and CRFK cells, and was independent of whether the cells expressed opioid receptors. The efflux of [3H][Dmt1]DALDA from Caco-2 cells was temperature dependent and was inhibited by DEPC, but was not affected by verapamil, an inhibitor of P-glycoprotein. These data show transcellular translocation of a highly polar 3+ charge tetrapeptide and suggest that [Dmt1]DALDA may not only distribute across the blood-brain barrier but also it may even have reasonable oral absorption. Zhao, K., Luo, G., Zhao, G-M., Schiller, P.W., and Szeto, H.H. Importance of Nonpharmacological Factors in Nicotine Self-administration. J. Pharmacol. Exp. Ther., 304, pp. 703-715, 2003.

Anabolic-Androgenic Steroids (AAS) & Aggression

In a recent study, Dr. Richard Melloni and his associates examined the effects of chronic AAS exposure during adolescent development on the arginine vasopressin (AVP) V1A receptor system regulating offensive aggression. They undertook this study because clinical and basic research have demonstrated a positive correlation between long-term use of AAS and negative behavioral effects including increased aggressive behavior. Studies also report increased hypothalamic AVP and facilitation of offensive aggression in male Syrian hamsters following repeated AAS exposure. Findings from this study show for the first time that exposure to high-dose of AAS during adolescent development can dramatically increase the binding of AVP V1A receptors in intact animals. These findings are significant as they demonstrate that increases in offensive aggression resulting from adolescent AAS exposure correlate directly with increases in AVP V1A receptor binding activity in several key areas of the hamster brain implicated in aggressive responding, but not in others. From a neuroanatomical standpoint, these data implicate enhanced AVP neural signaling via AVP V1A receptor in these aggression areas as potential neural substrates for adolescent AAS-facilitated offensive aggression. DeLeon, K.R., Grimes, J.M. and Melloni, Jr., R.H. Repeated Anabolic-Androgenic Steroids Treatment during Adolescence Increases Vasopressin V1A Receptor Binding in Syrian Hamsters: Correlation with Offensive Aggression, Hormones & Behavior, 42, pp. 182-191, 2002.

An Analysis of the Binding of Cocaine Analogues to the Monoamine Transporters Using Tensor Decomposition 3-D QSAR

The conformation and alignment of cocaine analogues bound to the monoamine transporter proteins were explored using the tensor decomposition 3-D QSAR method. It was proposed from these calculations that the bound conformation of these ligands to the three transporter proteins has the 3-beta aryl substituent in a conformation in which the aryl group is orthogonal or approximately orthogonal to the propane ring. Based upon these results, rigid and semi rigid tropane analogues were designed, synthesized, and their affinities for the monoamine transporters were determined. Appell, M., Dunn III, W.J., Reith, M.E.A., Miller, L. and Flippen-Anderson, J.L. An Analysis of the Binding of Cocaine Analogues to the Monoamine Transporters Using Tensor Decomposition 3-D QSAR. Bioorganic and Medicinal Chemistry, 10, pp. 1197-1206, 2002.

Opiates and HIV Infection: Role of Chemokines

The HIV virus attacks lymphocytes and macrophages by linking to a chemokine receptor, which introduces the virus into the cell. For several years, opiates have been shown to alter chemotaxis through inhibition of chemokine action and thereby modulate HIV function in vitro. This review clarifies how opiates can stimulate or inhibit this basic immune system. The analgesic property of opiates has been known since ancient times. Only recently has an appreciation of the broad effects of opioids on the inflammatory response emerged. Acting largely through mu-, kappa- and delta-opioid G protein-coupled receptors on T lymphocytes and macrophages, cognate ligands modulate many activities of these cells, including cytokine production. In addition to acting as chemotactic stimuli, opioids can, through the process of heterologous cross-desensitization, act as stop signals in leukocyte trafficking. When administered into the central nervous system, certain chemokines can cross-desensitize to the analgesic effect of opioids. Thus, opioids should be considered members of the cytokine family - future research on opioids could yield new therapies for inflammatory and infectious diseases, including HIV-1 infection. Rogers, T.J., Peterson, P.K. Opioid G Protein-coupled Receptors: Signals at the Crossroads of Inflammation. Trends Immunol., 24, pp. 116-121, 2003.

A related article explores the mechanism of this activity. Morphine alters the production of chemokines and the receptors to stimulate the increase in HIV infection. Injection drug use, remains a significant risk for acquiring HIV infection. The mechanisms by which morphine enhances HIV infection of human immune cells are largely unknown. This study was designed to determine the possible mechanisms by which morphine upregulates HIV infection of human blood monocyte-derived macrophages (MDM). Morphine significantly enhanced HIV R5 strain infection of MDM but had little effect on X4 strain infection. The macrophage-tropic R5 strain envelope-pseudotyped HIV infection was markedly increased by morphine, whereas murine leukemia virus envelope-pseudotyped HIV infection was not significantly affected. Furthermore, morphine significantly upregulated CCR5 receptor expression and inhibited the endogenous production of beta-chemokines in MDM. The opioid receptor antagonist naltrexone blocked the effects of morphine on the production of beta-chemokines. It is concluded that opiates enhance HIV R5 strain infection of macrophages through the downregulation of beta-chemokine production and upregulation of CCR5 receptor expression and may have an important role in HIV immunopathogenesis. Guo, C.J., Li, Y., Tian, S., Wang, X., Douglas, S.D., and Ho, W.Z. Morphine Enhances HIV Infection of Human Blood Mononuclear Phagocytes through Modulation of Beta-Chemokines and CCR5 Receptor. J. Investigative Med., 50, pp. 435-442, 2002.

In Vivo Activation of a Mutant µ-opioid Receptor by Antagonist: Future Direction for Opiate Pain Treatment Paradigm that Lacks Undesirable Side Effects

It had been previously discovered that :-opioid receptor in which serine 196 has been changed to an alanine (S196A), could be activated by receptor antagonists in vitro. That is to say that receptor antagonists such as naloxone and naltrexone caused the activation of G-protein coupled inward rectifying potassium channel 1 (GIRK1) when it was co-expressed in Xenopus oocytes with the mutant :-opioid receptor. In this study, investigators wanted to know whether this mutant receptor could be used to elicit the analgesic effects seen with opioids without the negative side effects such as dependence and tolerance. In order to do this, they introduced the mutant receptor into mice using a knock-in strategy and tested the analgesic effects. They found that in mice that have two copies of the mutant receptor, homozygous mutant mice, naloxone and naltrexone produced antinociceptive (painkiller) effects similar to those of partial agonists. In their evaluation of tolerance development, they found that the homozygous mutant mice treated chronically with morphine for 72 hours had a 37-fold increase in the 50% effective dose (ED50) of morphine. This is actually greater tolerance than is seen in wild type mice. However, tolerance to naltrexone was not observed in the homozygous mutant mice. When the investigators examined the development of dependence using agonist, morphine or naltrexone, they found that the homozygous mutant mice did develop some symptoms of dependence, but these were much less severe than what is seen in wild type animals and morphine. The ability of a receptor antagonist to elicit an antinociceptive effect without development of tolerance in these mutant mice represents an alternative approach to pain treatment paradigms. Law, P-Y., Yang, J.W., Guo, X., and Loh, H.H. In Vivo Activation of a Mutant _-opioid Receptor by Antagonist: Future Direction for Opiate Pain Treatment Paradigm that Lacks Undesirable Side Effects. Proc. Natl. Acad. Sci. U.S.A., 100(4), pp. 2117-2121, February 18, 2003.

Dimerization of Morphine and Orphanin FQ/Nociceptin Receptors: Generation of a Novel Opioid Receptor Subtype

Although orphanin FQ/nociceptin (OFQ/N) receptors are a member of the opioid receptor family of receptors, they bind traditional opioids with very poor affinity. In this study Dr. Pasternak and his research team demonstrate that mu opioid receptors can physically associate with OFQ/N receptors, resulting in a complex with a unique binding selectivity profile. Immunopre-cipitation of epitope-tagged OFQ/N receptors co-precipitates mu receptors. When the two receptors were co-expressed in CHO cells, [3H]OFQ/N retained its high binding affinity for its receptor. However, co-expression of the two receptors increased by up to 250-fold the affinity of a series of opioids in [3H]OFQ/N binding assays. This enhanced affinity was limited to agonists with high affinity for mu receptors. Selective kappa(1) and delta opioids did not lower binding. Despite the dramatic increase in affinity for the opioid agonists in co-expressing cells, the opioid antagonists naloxone and diprenorphine failed to compete [3H]OFQ/N binding. These findings suggest possible functionally significant interactions between OFQ/N and mu receptors, an observation consistent with the co-expression of mu and OFQ/N receptors in the dorsal horn of the spinal cord, the hippocampal formation, and the caudate/putamen and may help explain the reports of functional interactions between OFQ/N and opioids. Biochem. Biophys. Res. Commun. 297(3), pp. 659-663, September 27, 2002 Erratum in: Pan, Y.X., Bolan, E., and Pasternak, G.W. Dimerization of Morphine and Orphanin FQ/Nociceptin Receptors: Generation of a Novel Opioid Receptor Subtype. Biochem. Biophys. Res. Commun. 298(3), p. 456, November 1, 2002.

Genetic Dissociation of Opiate Tolerance and Physical Dependence in Delta-Opioid Receptor-1 and Preproenkephalin Knock-Out Mice

Previous experiments have shown that mice lacking a functional delta-opioid receptor (DOR-1) gene do not develop analgesic tolerance to morphine. Here Drs. Pintar and Pasternak and their colleagues report that mice lacking a functional gene for the endogenous ligand preproenkephalin (ppENK) show a similar tolerance deficit. In addition, they found that the DOR-1 and ppENK knock-outs as well as the NMDA receptor-deficient 129S6 inbred mouse strain, which also lacks tolerance, exhibit antagonist-induced opioid withdrawal. These data demonstrate that although signaling pathways involving ppENK, DOR, and NMDA receptor are necessary for the expression of morphine tolerance, other pathways independent of these factors can mediate physical dependence. Moreover, these studies illustrate that morphine tolerance can be genetically dissociated from physical dependence, and thus provide a genetic framework to assess more precisely the contribution of various cellular and molecular changes that accompany morphine administration to these processes. Nitsche, J.F., Schuller, A.G., King, M.A., Zengh, M., Pasternak, G.W., and Pintar, J.E. Genetic Dissociation of Opiate Tolerance and Physical Dependence in Delta-Opioid Receptor-1 and Preproenkephalin Knock-out Mice. J. Neurosci. 22(24), pp. 10906-10913, December 15, 2002.

Identification of a Signaling Network in Lateral Nucleus of Amygdala Important for Inhibiting Memory Specifically Related to Learned Fear

Post-traumatic stress disorder is a comorbid disorder that commonly occurs with substance abuse. Conditioned fear may be an excellent model for post-traumatic stress disorder and phobias. The lateral nucleus of the amygdala has been implicated as a central site within the brain mediating conditioned fear. Following fear conditioning the synaptic connections that feed into the lateral nucleus of the amygdala from the cortex and thalamus are greatly enhanced. In a recent paper that appeared in the December 2002 issue of Cell, Dr. Vladim Bolshakov, a NIDA grantee, in collaboration with Dr. Eric Kandel and his colleagues have identified gastrin releasing peptide and the gastrin releasing peptide receptor as important molecular mechanisms in this pathway regulating changes in synaptic strength in the lateral nucleus during fear conditioning. They report that gastrin releasing peptide is highly expressed in the lateral nucleus of the amygdala and in neurons that send projections into the lateral nucleus. When gastrin releasing peptide is secreted, the peptide acts on the gastrin releasing peptide receptors located on GABA inhibitory interneurons. The binding of the gastrin releasing peptide to its receptor excites GABA excitatory interneurons in the lateral nucleus. Mice lacking the gastrin releasing peptide receptor showed that tonic inhibition in the lateral nucleus was decreased while long term potentiation (LTP) of the cortico-amygdala pathway is enhanced. Long term memory for fear to both contextual and auditory cues was found to be increased. The enhancement of long-term memory is specific to fear conditioning because animals showed normal amounts of anxiety in behavioral tests that examine anxiety in mice and the mice showed no deficits on spatial memory tasks. This work demonstrates the potential of gastrin releasing peptide and receptor as important therapeutic targets for the treatment of phobia and post-traumatic stress disorder. Shumyatsky, G.P., Tsvetkov, E., Malleret, G., Vronskayaa, S., Hatton, M., Hampton, L., Battey, J., Dulac, C., Kandel, E., and Bolshakov, V. Identification of a Signaling Network in Lateral Nucleus of Amygdala Important for Inhibiting Memory Specifically Related to Learned Fear, Cell. 11(6), pp. 905-918, December 13, 2002.

Active Heroin Administration Induces Specific Genomic Responses in the Nucleus Accumbens Shell

The shell of the nucleus accumbens (NAc) is a brain region important for reward by mediating retrieval of learned associations affecting goal-directed behavior. Dr. Anton N.M. Schoffelmeer and his colleagues studied the genomic response in the rat NAc after long-term withdrawal of active and passive heroin administration. Using different cohorts of rats with a history of heroin or cocaine exposure, they validated and examined a set of 40 transcripts that is down-regulated in the NAc shell after long-term cessation of heroin self-administration by real-time quantitative PCR. They found that active, but not passive administration induces long-term genomic responses in the NAc shell. These down-regulated transcripts comprise a large functional diversity of proteins involved in basic cellular processes such as neuronal growth, apoptosis, signal transduction, neuropeptide synthesis and release, transcription, translation, and cell metabolism. These data indicate that cellular processes are still affected long after extinction of heroin self-administration. Combined with the integral role of the NAc shell with the limbic system, the authors suggest that long-lasting changes in cellular functional may change synaptic transmission in related brain structures. They also point out that these genomic changes outlast simple pharmacological effects of the drugs themselves. Taken together, drug-seeking behavior may be a direct result of altered genetic and cellular networks. Jacobs, E.H., Spijker, S., Verhoog, C.W., Kamprath, K., DeVries, T.J., Smit, A.B., and Schoffelmeer, A.N.M. Active Heroin Administration Induces Specific Genomic Responses in the Nucleus Accumbens Shell. FASEB J, 16(14), pp. 1961-1963, 2002.

Guiding GABAergic Neurons

Understanding the cellular and molecular consequences of prenatal drug exposure requires a foundation in developmental neurobiology. During drug exposure, the normal differentiation and positioning of neurons can be altered in a myriad of ways. Still, NIDA-sponsored research is dissecting out the complexities of these processes. A substantial number of cortical GABAergic interneurons are born in the basal telencephalon and migrate tangentially to reach their final destination in the neocortex and hippocampus. To accomplish this, they must interpret multiple guidance cues encountered through their trajectory. Previous work had demonstrated that the basal telencephalon contains a repulsive activity for tangentially migrating cells, whereas the cerebral cortex contains an attractive activity. While the repulsive membrane proteins Slit1, Slit2, and Netrin1 are all good candidates to act as repulsive cues in the basal telecephalon, whether they are necessary for the entirety of repulsive cues was unknown. Using a combination of explants and Slit1, Slit2 and Netrin mutant mice, the lab of John Rubenstein was able to address this issue directly. In brief, their experiments demonstrate that whereas Slit1 and Slit2 are not necessary for tangential migration of interneurons to the cortex, these proteins regulate neuronal migration within the basal telencephalon by controlling cell positioning close to the midline. As such, these results argue that both attractive and repulsive activities direct interneuron migration to the cortex, and that distinct factors control distinct choice points along the way. As a result, a complex group of guidance cues are likely to be vitally involved in GABAergic development. Marin, O., Plump, A., Flames, N., Sanchez-Camacho, C., Tessier-Lavigne, M., and Rubenstein, J. Directional Guidance of Interneuron Migration to the Cerebral Cortex Relies on Subcortical Slit1/2-Independent Repulsion and Cortical Attraction. Development, 130, pp. 1889-1901, 2003.