Paul J. Gresch

Hippocampal interneurons are abnormal in schizophrenia

OBJECTIVE The cellular substrate of hippocampal dysfunction in schizophrenia remains unknown. We tested the hypothesis that hippocampal interneurons are abnormal in schizophrenia, but that the total number of hippocampal neurons in the pyramidal cell layer is normal. METHODS We collected whole hippocampal specimens of 13 subjects with schizophrenia and 20 matched healthy control subjects to study the number of all neurons, the somal volume of neurons, the number of somatostatin- and parvalbumin-positive interneurons and the messenger RNA levels of somatostatin, parvalbumin and glutamic acid decarboxylase 67. RESULTS The total number of hippocampal neurons in the pyramidal cell layer was normal in schizophrenia, but the number of somatostatin- and parvalbumin-positive interneurons, and the level of somatostatin, parvalbumin and glutamic acid decarboxylase mRNA expression were reduced. CONCLUSIONS The study provides strong evidence for a specific defect of hippocampal interneurons in schizophrenia and has implications for emerging models of hippocampal dysfunction in schizophrenia.

Dysregulation of the norepinephrine transporter sustains cortical hypodopaminergia and schizophrenia-like behaviors in neuronal rictor null mice.

A novel animal model highlights the link between Akt dysfunction, reduced cortical dopamine function, norepinephrine transporters, and schizophrenia-like behaviors.

Hippocampal Interneurons in Bipolar Disorder

CONTEXT Postmortem studies have reported decreased density and decreased gene expression of hippocampal interneurons in bipolar disorder, but neuroimaging studies of hippocampal volume and function have been inconclusive. OBJECTIVE To assess hippocampal volume, neuron number, and interneurons in the same specimens of subjects with bipolar disorder and healthy control subjects. DESIGN Whole human hippocampi of 14 subjects with bipolar disorder and 18 healthy control subjects were cut at 2.5-mm intervals and sections from each tissue block were either Nissl-stained or stained with antibodies against somatostatin or parvalbumin. Messenger RNA was extracted from fixed tissue and real-time quantitative polymerase chain reaction was performed. SETTING Basic research laboratories at Vanderbilt University and McLean Hospital. SAMPLES Brain specimens from the Harvard Brain Tissue Resource Center at McLean Hospital. MAIN OUTCOME MEASURES Volume of pyramidal and nonpyramidal cell layers, overall neuron number and size, number of somatostatin- and parvalbumin-positive interneurons, and messenger RNA levels of somatostatin, parvalbumin, and glutamic acid decarboxylase 1. RESULTS The 2 groups did not differ in the total number of hippocampal neurons, but the bipolar disorder group showed reduced volume of the nonpyramidal cell layers, reduced somal volume in cornu ammonis sector 2/3, reduced number of somatostatin- and parvalbumin-positive neurons, and reduced messenger RNA levels for somatostatin, parvalbumin, and glutamic acid decarboxylase 1. CONCLUSION Our results indicate a specific alteration of hippocampal interneurons in bipolar disorder, likely resulting in hippocampal dysfunction.

5-Hydroxytryptamine2C receptor contribution to m-chlorophenylpiperazine and N-methyl-beta-carboline-3-carboxamide-induced anxiety-like behavior and limbic brain activation.

Activation of 5-hydroxytryptamine2C (5-HT2C) receptors by the 5-HT2 receptor agonist m-chlorophenylpiperazine (m-CPP) elicits anxiety in humans and anxiety-like behavior in animals. We compared the effects of m-CPP with the anxiogenic GABAA receptor inverse agonist N-methyl-β-carboline-3-carboxamide (FG-7142) on both anxiety-like behavior and regional brain activation using functional magnetic resonance imaging (fMRI) in the rat. We also determined whether the selective 5-HT2C receptor antagonist SB 242084 [6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride] would blunt m-CPP or FG-7142-induced neuronal activation. Both m-CPP (3 mg/kg i.p.) and FG-7142 (10 mg/kg i.p.) elicited anxiety-like behavior when measured in the social interaction test, and pretreatment with SB 242084 (1 mg/kg i.p.) completely blocked the behavioral effects of both anxiogenic drugs. Regional brain activation in vivo in response to anxiogenic drug challenge was determined by blood oxygen level-dependent (BOLD) fMRI using a powerful 9.4T magnet. Region of interest analyses revealed that m-CPP and FG-7142 significantly increased BOLD signals in brain regions that have been linked to anxiety, including the amygdala, dorsal hippocampus, and medial hypothalamus. These BOLD signal increases were blocked by pretreatment with SB 242084. In contrast, injection of m-CPP and FG-7142 resulted in BOLD signal decreases in the medial prefrontal cortex that were not blocked by SB 242084. In conclusion, the brain activation signals produced by anxiogenic doses of both m-CPP and FG-7142 are mediated at least partially by the 5-HT2C receptor, indicating that this receptor is a key component in anxiogenic neural circuitry.

The serotonin 2C receptor potently modulates the head-twitch response in mice induced by a phenethylamine hallucinogen

RationaleHallucinogenic serotonin 2A (5-HT2A) receptor partial agonists, such as (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT2A receptor antagonists. In addition to 5-HT2A receptors, DOI and most other serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT2C receptors.ObjectivesWe tested for involvement of 5-HT2C receptors in the HTR induced by DOI.ResultsComparison of 5-HT2C receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT2C receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT2A receptors in frontal cortex explained the strain difference, including 5-HT2A receptor density, Gαq or Gαi/o protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT2C receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT2C receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI.ConclusionsWe conclude that the HTR to DOI in mice is strongly modulated by 5-HT2C receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT2 receptors.

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter

Serotonin [i.e., 5-hydroxytryptamine (5-HT)]–targeted antidepressants are in wide use for the treatment of mood disorders, although many patients do not show a response or experience unpleasant side effects. Psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (i.e., “ecstasy”), also impact 5-HT signaling. To help dissect the contribution of 5-HT signaling to the actions of these and other agents, we developed transgenic mice in which high-affinity recognition of multiple antidepressants and cocaine is eliminated. Our animals possess a modified copy of the 5-HT transporter (i.e., SERT, slc6a4) that bears a single amino acid substitution, I172M, proximal to the 5-HT binding site. Although the M172 substitution does not impact the recognition of 5-HT, this mutation disrupts high-affinity binding of many competitive antagonists in transfected cells. Here, we demonstrate that, in M172 knock-in mice, basal SERT protein levels, 5-HT transport rates, and 5-HT levels are normal. However, SERT M172 mice display a substantial loss of sensitivity to the selective 5-HT reuptake inhibitors fluoxetine and citalopram, as well as to cocaine. Through a series of biochemical, electrophysiological, and behavioral assays, we demonstrate the unique properties of this model and establish directly that SERT is the sole protein responsible for selective 5-HT reuptake inhibitor-mediated alterations in 5-HT clearance, in 5-HT1A autoreceptor modulation of raphe neuron firing, and in behaviors used to predict the utility of antidepressants.

Behavioral Tolerance to Lysergic Acid Diethylamide is Associated with Reduced Serotonin-2A Receptor Signaling in Rat Cortex

Tolerance is defined as a decrease in responsiveness to a drug after repeated administration. Tolerance to the behavioral effects of hallucinogens occurs in humans and animals. In this study, we used drug discrimination to establish a behavioral model of lysergic acid diethylamide (LSD) tolerance and examined whether tolerance to the stimulus properties of LSD is related to altered serotonin receptor signaling. Rats were trained to discriminate 60 μg/kg LSD from saline in a two-lever drug discrimination paradigm. Two groups of animals were assigned to either chronic saline treatment or chronic LSD treatment. For chronic treatment, rats from each group were injected once per day with either 130 μg/kg LSD or saline for 5 days. Rats were tested for their ability to discriminate either saline or 60 μg/kg LSD, 24 h after the last chronic injection. Rats receiving chronic LSD showed a 44% reduction in LSD lever selection, while rats receiving chronic vehicle showed no change in percent choice on the LSD lever. In another group of rats receiving the identical chronic LSD treatment, LSD-stimulated [35S]GTPγS binding, an index of G-protein coupling, was measured in the rat brain by autoradiography. After chronic LSD, a significant reduction in LSD-stimulated [35S]GTPγS binding was observed in the medial prefrontal cortex and anterior cingulate cortex. Furthermore, chronic LSD produced a significant reduction in 2,5-dimethoxy-4-iodoamphetamine-stimulated [35S]GTPγS binding in medial prefrontal cortex and anterior cingulate cortex, which was blocked by MDL 100907, a selective 5-HT2A receptor antagonist, but not SB206553, a 5-HT2C receptor antagonist, indicating a reduction in 5-HT2A receptor signaling. 125I-LSD binding to 5-HT2A receptors was reduced in cortical regions, demonstrating a reduction in 5-HT2A receptor density. Taken together, these results indicate that adaptive changes in LSD-stimulated serotonin receptor signaling may mediate tolerance to the discriminative stimulus effects of LSD.

The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

Significance Dopamine (DA) signaling provides important, modulatory control of movement, at tention, and reward. Disorders linked to changes in DA signaling include Parkinson’s disease, attention-deficit hyperactivity disorder, schizophrenia, autism spectrum disorder, and addiction. We identified multiple, functional polymorphisms in the human DA transporter (DAT) gene and showed that one of these variants, which produces the amino acid substitution Val559 (wild-type DATs express Ala559), exhibits normal DA uptake accompanied by a spontaneous outward efflux of the neurotransmitter, reminiscent of the actions of the psychostimulant amphetamine. Here, we identify multiple biochemical, physiological, and behavioral perturbations that arise from DAT Val559 expression in vivo, supporting spontaneous DA efflux as a heretofore-unrecognized mechanism that may underlie multiple DA-linked neurobehavioral disorders. Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness.

5-Hydroxytryptamine (Serotonin)2A Receptors in Rat Anterior Cingulate Cortex Mediate the Discriminative Stimulus Properties of d-Lysergic Acid Diethylamide

d-Lysergic acid diethylamide (LSD), an indoleamine hallucinogen, produces profound alterations in mood, thought, and perception in humans. The brain site(s) that mediates the effects of LSD is currently unknown. In this study, we combine the drug discrimination paradigm with intracerebral microinjections to investigate the anatomical localization of the discriminative stimulus of LSD in rats. Based on our previous findings, we targeted the anterior cingulate cortex (ACC) to test its involvement in mediating the discriminative stimulus properties of LSD. Rats were trained to discriminate systemically administered LSD (0.085 mg/kg s.c.) from saline. Following acquisition of the discrimination, bilateral cannulae were implanted into the ACC (AP, +1.2 mm; ML, ±1.0 mm; DV, –2.0 mm relative to bregma). Rats were tested for their ability to discriminate varying doses of locally infused LSD (0.1875, 0.375, and 0.75 μg/side) or artificial cerebrospinal fluid (n = 3–7). LSD locally infused into ACC dose-dependently substituted for systemically administered LSD, with 0.75 μg/side LSD substituting completely (89% correct). Systemic administration of the selective 5-hydroxytryptamine (serotonin) (5-HT)2A receptor antagonist R-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (M100907; 0.4 mg/kg) blocked the discriminative cue of LSD (0.375 μg/side) infused into ACC (from 68 to 16% drug lever responding). Furthermore, M100907 (0.5 μg/μl/side) locally infused into ACC completely blocked the stimulus effects of systemic LSD (0.04 mg/kg; from 80 to 12% on the LSD lever). Taken together, these data indicate that 5-HT2A receptors in the ACC are a primary target mediating the discriminative stimulus properties of LSD.

Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake

The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre‐mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full‐length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full‐length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full‐length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro‐opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild‐type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood–brain barrier and its systemic delivery reduced food intake in wild‐type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre‐mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader–Willi syndrome.