Rebecca Ahlbrand

Risperidone pretreatment prevents elevated locomotor activity following neonatal hippocampal lesions.

Long-standing behavioral abnormalities emerge after puberty in rats following neonatal hippocampal lesion, providing a developmental model of abnormal rat behavior that may have predictive validity in identifying compounds effective in treating symptoms of schizophrenia. We sought to test the predictive validity of the neonatal hippocampal lesion model in identifying preventive treatment for first-episode psychosis. We determined the effect of risperidone, recently studied for prevention of first-episode psychosis, on the development of elevated locomotor activity following neonatal hippocampal lesions. Rat pups received hippocampal or sham lesions on postnatal day 7, followed by treatment with risperidone or vehicle from postnatal days 35 to 56. Locomotor activity in response to novelty, amphetamine, and nocturnal locomotion were determined on postnatal day 57. Low-dose risperidone (45 μg/kg) pretreatment prevented elevated locomotor activity in some, but not all, of the behavioral tasks following neonatal hippocampal lesions. In contrast, higher risperidone pretreatment was less effective in preventing elevated locomotor activity following neonatal hippocampal lesions. Because low risperidone dosages were also found to be effective in preventing first-episode psychosis in human studies, these data support the predictive validity of the hippocampal lesion model in identifying medications for prevention of first-episode psychosis. Additionally, these data support the use of low-dose risperidone in psychosis prevention, and suggest the possibility that higher risperidone doses could be less effective in this application.

Individual differences in maternal response to immune challenge predict offspring behavior: Contribution of environmental factors

Maternal infection during pregnancy elevates risk for schizophrenia and related disorders in offspring. Converging evidence suggests the maternal inflammatory response mediates the interaction between maternal infection, altered brain development, and behavioral outcome. The extent to which individual differences in the maternal response to immune challenge influence the development of these abnormalities is unknown. The present study investigated the impact of individual differences in maternal response to the viral mimic polyinosinic:polycytidylic acid (poly I:C) on offspring behavior. We observed significant variability in body weight alterations of pregnant rats induced by administration of poly I:C on gestational day 14. Furthermore, the presence or absence of maternal weight loss predicted MK-801 and amphetamine stimulated locomotor abnormalities in offspring. MK-801 stimulated locomotion was altered in offspring of all poly I:C treated dams; however, the presence or absence of maternal weight loss resulted in decreased and modestly increased locomotion, respectively. Adult offspring of poly I:C treated dams that lost weight exhibited significantly decreased amphetamine stimulated locomotion, while offspring of poly I:C treated dams without weight loss performed similarly to vehicle controls. Social isolation and increased maternal age predicted weight loss in response to poly I:C but not vehicle injection. In combination, these data identify environmental factors associated with the maternal response to immune challenge and functional outcome of offspring exposed to maternal immune activation.

Effect of paliperidone and risperidone on extracellular glutamate in the prefrontal cortex of rats exposed to prenatal immune activation or MK-801

The NMDA glutamate hypofunction model of schizophrenia is based in part upon acute effects of NMDA receptor blockade in humans and rodents. Several laboratories have reported glutamate system abnormalities following prenatal exposure to immune challenge, a known environmental risk factor for schizophrenia. Here we report indices of NMDA glutamate receptor hypofunction following prenatal immune activation, as well as the effects of treatment during periadolescence with the atypical antipsychotic medications risperidone and paliperidone. Pregnant Sprague-Dawley rats were injected with polyinosinic:polycytidylic acid (poly I:C) or saline on gestational day 14. Male offspring were treated orally via drinking water with vehicle, risperidone (0.01mg/kg/day), or paliperidone (0.01mg/kg/day) between postnatal days 35 and 56 (periadolescence) and extracellular glutamate levels in the prefrontal cortex were determined by microdialysis at PD 56. Consistent with decreased NMDA receptor function, MK-801-induced increases in extracellular glutamate concentration were markedly blunted following prenatal immune activation. Further suggesting NMDA receptor hypofunction, prefrontal cortex basal extracellular glutamate was significantly elevated (p<0.05) in offspring of poly I:C treated dams. Pretreatment with low dose paliperidone or risperidone (0.01mg/kg/day postnatal days 35-56) normalized prefrontal cortical basal extracellular glutamate (p<0.05 vs. poly I:C vehicle-treatment). Pretreatment with paliperidone and risperidone also prevented the acute MK-801-induced increase in extracellular glutamate. These observations demonstrate decreased NMDA receptor function and elevated extracellular glutamate, two key features of the NMDA glutamate receptor hypofunction model of schizophrenia, during periadolescence following prenatal immune activation. Treatment with the atypical antipsychotic medications paliperidone and risperidone normalized basal extracellular glutamate. Demonstration of glutamatergic abnormalities consistent with the NMDA glutamate receptor hypofunction model of schizophrenia as an early developmental consequence of prenatal immune action provides a model to identify novel early interventions targeting glutamatergic systems which play an important role in both positive and negative symptoms of schizophrenia.

Expression of the glucocorticoid-induced receptor mRNA in rat brain

The glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor awaiting pharmacological characterization. GIR was originally identified in murine thymoma cells, and shows a widespread, yet not completely complementary distribution in mouse and human brain. Expression of the mouse GIR gene is modulated by dexamethasone in the brain and periphery, suggesting that GIR function is directly responsive to glucocorticoid signals. The rat GIR was cloned from rat prefrontal cortex by our group and was shown to be up-regulated following chronic amphetamine. The physiological role of GIR in the rat is not known at present. In order to gain a clearer understanding of the potential functions of GIR in the rat, we performed a detailed mapping of GIR mRNA expression in the rat brain. GIR mRNA showed widespread distribution in forebrain limbic and thalamic structures, and a more restricted distribution in hindbrain areas such as the spinal trigeminal nucleus and the median raphe nucleus. Areas with moderate to high levels of GIR include olfactory regions such as the nucleus of olfactory tract, hippocampus, various thalamic nuclei, cortical layers, and some hypothalamic nuclei. In comparison with previous studies, significant regional differences exist in GIR distribution in mouse and rat brain, particularly in the thalamus, striatum and in hippocampus at a cellular level. Overall, the expression of GIR in rat brain more closely approaches that seen previously in human than mouse, suggesting that rat models may be more informative for understanding the role of GIR in glucocorticoid physiology and glucocorticoid-related disease states. GIR mRNA distribution in the rat indicates a potential role of this receptor in the control of feeding and ingestive behavior, regulation of stress and emotional behavior, learning and memory, and, drug reinforcement and reward.

C57BL/6J MICE EXHIBIT REDUCED DOPAMINE D3 RECEPTOR-MEDIATED LOCOMOTOR-INHIBITORY FUNCTION RELATIVE TO DBA/2J MICE

Previous reports have identified greater sensitivity to the locomotor-stimulating, sensitizing, and reinforcing effects of amphetamine in inbred C57BL/6J mice relative to inbred DBA/2J mice. The dopamine D3 receptor (D3R) plays an inhibitory role in the regulation of rodent locomotor activity, and exerts inhibitory opposition to D1 receptor (D1R)-mediated signaling. Based on these observations, we investigated D3R expression and D3R-mediated locomotor-inhibitory function, as well as D1R binding and D1R-mediated locomotor-stimulating function, in C57BL/6J and DBA/2J mice. C57BL/6J mice exhibited lower D3R binding density (-32%) in the ventral striatum (nucleus accumbens/islands of Calleja), lower D3R mRNA expression (-26%) in the substantia nigra/ventral tegmentum, and greater D3R mRNA expression (+40%) in the hippocampus, relative to DBA/2J mice. There were no strain differences in DR3 mRNA expression in the ventral striatum or prefrontal cortex, nor were there differences in D1R binding in the ventral striatum. Behaviorally, C57BL/6J mice were less sensitive to the locomotor-inhibitory effect of the D3R agonist PD128907 (10 microg/kg), and more sensitive to the locomotor-stimulating effects of novelty, amphetamine (1 mg/kg), and the D1R-like agonist +/- -1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8,-diol hydrochloride (SKF38393) (5-20 mg/kg) than DBA/2J mice. While the selective D3R antagonist N-(4-[4-{2,3-dichlorphenyl}-1 piperazinyl]butyl)-2-fluorenylcarboxamide (NGB 2904) (0.01-1.0 mg/kg) augmented novelty-, amphetamine-, and SKF38393-induced locomotor activity in DBA/2J mice, it reduced novelty-induced locomotor activity in C57BL/6J mice. Collectively, these results demonstrate that C57BL/6J mice exhibit less D3R-mediated inhibitory function relative to DBA/2J mice, and suggest that reduced D3R-mediated inhibitory function may contribute to heightened sensitivity to the locomotor-stimulating effects of amphetamine in the C57BL/6J mouse strain. Furthermore, these data demonstrate that comparisons between C57BL/6J and DBA/2J mouse strains provide a model for elucidating the molecular determinants of genetic influence on D3R function.

Relative expression of D3 dopamine receptor and alternative splice variant D3nf mRNA in high and low responders to novelty.

Studies in rodents suggest an important role for the D3 dopamine receptor in regulating locomotor responses to spatial novelty and psychostimulants. The D3 receptor alternatively spliced variant D3nf produces a non-dopamine binding protein that may alter D3 receptor localization by dimerizing with the full-length receptor. In the high responder/low responder (HR/LR) model, the locomotor response to an inescapable, novel spatial environment predicts individual differences in the locomotor and rewarding effects of psychostimulants. We hypothesized that individual differences in D3 receptor expression could contribute to individual differences in the locomotor response to novelty in the HR/LR model. To test this hypothesis, we screened rats for response to a novel spatial environment and analyzed brain tissue for mRNA levels of the D3 receptor and D3nf by real-time RT-PCR. The ratios of D3/D3nf mRNA in prefrontal cortex and substantia nigra/ventral tegmentum were significantly lower in HRs than in LRs. There were no differences in relative expression of D3/D3nf between HRs and LRs in nucleus accumbens. These data further support a role for the D3 dopamine receptor in behavioral responses to novelty and, given the established relationship between novelty and psychostimulant responses, suggest that the D3 receptor may be an important target for assessment of drug abuse vulnerability. Additionally, these findings are consistent with the hypothesis that alternative splicing may contribute to regulation of D3 dopamine receptor function.

Evidence for involvement of nitric oxide and GABAB receptors in MK-801- stimulated release of glutamate in rat prefrontal cortex

Systemic administration of NMDA receptor antagonists elevates extracellular glutamate within prefrontal cortex. The cognitive and behavioral effects of NMDA receptor blockade have direct relevance to symptoms of schizophrenia, and recent studies demonstrate an important role for nitric oxide and GABA(B) receptors in mediating the effects of NMDA receptor blockade on these behaviors. We sought to extend those observations by directly measuring the effects of nitric oxide and GABA(B) receptor mechanisms on MK-801-induced glutamate release in the prefrontal cortex. Systemic MK-801 injection (0.3 mg/kg) to male Sprague-Dawley rats significantly increased extracellular glutamate levels in prefrontal cortex, as determined by microdialysis. This effect was blocked by pre-treatment with the nitric oxide synthase inhibitor L-NAME (60 mg/kg). Reverse dialysis of the nitric oxide donor SNAP (0.5-5 mM) directly into prefrontal cortex mimicked the effect of systemic MK-801, dose-dependently elevating cortical extracellular glutamate. The effect of MK-801 was also blocked by systemic treatment with the GABA(B) receptor agonist baclofen (5 mg/kg). In combination, these data suggest increased nitric oxide formation is necessary for NMDA antagonist-induced elevations of extracellular glutamate in the prefrontal cortex. Additionally, the data suggest GABA(B) receptor activation can modulate the NMDA antagonist-induced increase in cortical glutamate release.

Microglial Acid Sensing Regulates Carbon Dioxide-Evoked Fear

BACKGROUND Carbon dioxide (CO2) inhalation, a biological challenge and pathologic marker in panic disorder, evokes intense fear and panic attacks in susceptible individuals. The molecular identity and anatomic location of CO2-sensing systems that translate CO2-evoked fear remain unclear. We investigated contributions of microglial acid sensor T cell death-associated gene-8 (TDAG8) and microglial proinflammatory responses in CO2-evoked behavioral and physiological responses. METHODS CO2-evoked freezing, autonomic, and respiratory responses were assessed in TDAG8-deficient ((-/-)) and wild-type ((+/+)) mice. Involvement of TDAG8-dependent microglial activation and proinflammatory cytokine interleukin (IL)-1β with CO2-evoked responses was investigated using microglial blocker, minocycline, and IL-1β antagonist IL-1RA. CO2-chemosensitive firing responses using single-cell patch clamping were measured in TDAG8(-/-) and TDAG8(+/+) mice to gain functional insights. RESULTS TDAG8 expression was localized in microglia enriched within the sensory circumventricular organs. TDAG8(-/-) mice displayed attenuated CO2-evoked freezing and sympathetic responses. TDAG8 deficiency was associated with reduced microglial activation and proinflammatory cytokine IL-1β within the subfornical organ. Central infusion of microglial activation blocker minocycline and IL-1β antagonist IL-1RA attenuated CO2-evoked freezing. Finally, CO2-evoked neuronal firing in patch-clamped subfornical organ neurons was dependent on acid sensor TDAG8 and IL-1β. CONCLUSIONS Our data identify TDAG8-dependent microglial acid sensing as a unique chemosensor for detecting and translating hypercapnia to fear-associated behavioral and physiological responses, providing a novel mechanism for homeostatic threat detection of relevance to psychiatric conditions such as panic disorder.

Fluoxetine and aripiprazole treatment following prenatal immune activation exert longstanding effects on rat locomotor response

AIM Studies characterizing treatment interventions in a naturalistic setting suggest that antidepressant and antipsychotic medications may be equally effective in improving clinical outcome in individuals at high risk for first-episode psychosis. Of interest, both beneficial as well as potentially adverse effects have been observed following fluoxetine treatment in a mouse prenatal immune activation model of relevance to psychosis prevention. We sought to extend those findings by examining the effects of fluoxetine, as well as the antipsychotic medication aripiprazole, in a rat prenatal immune activation model. METHODS Pregnant Sprague-Dawley rats were injected with poly I:C or saline on gestational day 14. Offspring of poly I:C and saline-treated dams received fluoxetine (10.0 mg/kg/d), aripiprazole (0.66 mg/kg/d), or vehicle from postnatal days 35 to 70. Locomotor responses to novelty, saline injection, and amphetamine (1 and 5 mg/kg) were determined at three months, i.e., 21 days following drug discontinuation. RESULTS Both fluoxetine and aripiprazole had beneficial effects on behavioral response to amphetamine (1 mg/kg) at 3 months, ameliorating the impact of prenatal immune activation on offspring of poly I:C-treated dams. Significantly, both drugs also exerted effects in offspring of control (saline-treated) dams on locomotor response to injection. CONCLUSIONS Fluoxetine and aripiprazole pretreatment of poly I:C offspring from postnatal days 35 to 70 stabilized response to amphetamine exposure persisting through 3 months of age, similar to earlier findings in mice that fluoxetine treatment following prenatal immune activation prevented altered locomotor response to amphetamine. The current data also confirm earlier findings of potential adverse behavioral effects in offspring of control dams following treatment with fluoxetine and antipsychotic medications, highlighting the potential for both therapeutic as well as safety concerns with exposure to preventive pharmacological treatments over the course of adolescent development. Further study is needed to determine clinical and epidemiological consequences of these pre-clinical findings.

Neuropeptide Y Impairs Retrieval of Extinguished Fear and Modulates Excitability of Neurons in the Infralimbic Prefrontal Cortex

Neuropeptide Y (NPY), a 36 aa peptide, regulates stress and emotional behaviors. Preclinical and clinical studies support an association of NPY with trauma-evoked syndromes such as posttraumatic stress disorder (PTSD), although the exact contribution of NPY is not clear. In the current study, we examined functional attributes of NPY in the infralimbic (IL) cortex, an area that regulates fear memories and is reported to be hypoactive in PTSD. Carriers of NPY gene polymorphism rs16147 have been reported to have elevated prefrontal NPY expression. Infusion of NPY into the IL cortex in rats significantly impaired fear extinction memory without affecting conditioned fear expression or acquisition of extinction. Neuroendocrine stress response, depression-like behavior, and working memory performance were not affected by NPY infusion into the IL. The NPY Y1 receptor antagonist BIBO3304 completely abolished NPY effects on fear extinction retrieval. Y1 receptor expression was localized on CaMKII-positive pyramidal projection neurons and GAD67-positive interneurons in the IL. Patch-clamp recordings revealed increased inhibitory synaptic transmission onto IL projection neurons in the presence of NPY. Thus, NPY dampens excitability of IL projection neurons and impairs retrieval of extinction memory by inhibiting consolidation of extinction. Of relevance to PTSD, elevation of prefrontal NPY attributable to the genetic polymorphism rs16147 may contribute to IL hypoactivity, resulting in impaired extinction memory and susceptibility to the disorder. SIGNIFICANCE STATEMENT Neuropeptide Y (NPY), a stress modulatory transmitter, is associated with posttraumatic stress disorder (PTSD). Contribution of NPY to PTSD symptomology is unclear. PTSD patients have reduced activity in the infralimbic (IL) subdivision of the medial prefrontal cortex (mPFC), associated with compromised extinction memory. No information exists on fear modulation by NPY in the IL cortex, although NPY and NPY receptors are abundant in these areas. This study shows that IL NPY inhibits consolidation of extinction, resulting in impaired retrieval of extinction memory and modulates excitability of IL projection neurons. In addition to providing a novel perspective on extinction memory modulation by NPY, our findings suggest that elevated mPFC NPY in gene polymorphism rs16147 carriers or after chronic stress could increase susceptibility to PTSD.