A novel role for the ADHD risk gene latrophilin-3 in learning and memory in Lphn3 knockout rats.

Latrophilins (LPHNs) are adhesion G protein-coupled receptors with three isoforms but only LPHN3 is brain specific (caudate, prefrontal cortex, dentate, amygdala, and cerebellum). Variants of LPHN3 are associated with ADHD. Null mutations of Lphn3 in rat, mouse, zebrafish, and Drosophila result in hyperactivity, but its role in learning and memory (L&M) is largely unknown. Using our Lphn3 knockout (KO) rats we examined the cognitive abilities, long-term potentiation (LTP) in CA1, NMDA receptor expression, and neurohistology from heterozygous breeding pairs. KO rats were impaired in egocentric L&M in the Cincinnati water maze, spatial L&M and cognitive flexibility in the Morris water maze (MWM), with no effects on conditioned freezing, novel object recognition, or temporal order recognition. KO-associated locomotor hyperactivity had no effect on swim speed. KO rats had reduced early-LTP but not late-LTP and had reduced hippocampal NMDA-NR1 expression. In a second experiment, KO rats responded to a light prepulse prior to an acoustic startle pulse, reflecting visual signal detection. In a third experiment, KO rats given extra MWM pretraining and hidden platform overtraining showed no evidence of reaching WT rats' levels of learning. Nissl histology revealed no structural abnormalities in KO rats. LPHN3 has a selective effect on egocentric and allocentric L&M without effects on conditioned freezing or recognition memory.

Knockout of latrophilin-3 in Sprague-Dawley rats causes hyperactivity, hyper-reactivity, under-response to amphetamine, and disrupted dopamine markers.

Attention deficit hyperactivity disorder is a pervasive developmental disorder characterized by inattention, impulsivity, and hyperactivity and is 75-90% heritable. Latrophilin-3 (LPHN3; or ADGRL(3)) is associated with a subtype of ADHD, but how it translates to symptoms is unknown. LPHN3 is a synaptic adhesion G protein coupled receptor that binds to fibronectin leucine rich transmembrane protein 3 and teneurin-3 (FLRT3 and TEN-3). We created a null mutation of Lphn3 (KO) in Sprague-Dawley rats using CRISPR/Cas9 to delete exon-3. The KO rats had no effects on reproduction or survival but reduced growth. KO females showed catch-up weight gain whereas KO males did not. We tested WT and KO littermates for home-cage activity, anxiety-like behavior, acoustic startle response, and activity after amphetamine challenge. Expression of Lphn3-related genes, monoamines, and receptors were determined. Lphn3 KO rats showed persistent hyperactivity, increased acoustic startle, reduced activity in response to amphetamine relative to baseline, and female-specific reduced anxiety-like behavior. Expression of Lphn1, Lphn2, and Flrt3 by qPCR and their protein products by western-blot analysis showed no compensatory upregulation. Striatal tyrosine hydroxylase, aromatic L-amino acid decarboxylase (AADC), and the dopamine transporter were increased and dopamine D1 receptor (DRD1) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) decreased with no changes in DRD2, DRD4, vesicular monoamine transporter-2, N-methyl-d-aspartate (NMDA)-NR1, -NR2A, or -NR2B. LPHN3 is expressed in many brain regions but its function is largely unknown. Data from human, mouse, zebrafish, Drosophila and our new Lphn3 KO rat data collectively show that its disruption is significantly correlated with hyperactivity and associated striatal changes in dopamine markers.

Effects of Preweaning Manganese in Combination with Adult Striatal Dopamine Lesions on Monoamines, BDNF, TrkB, and Cognitive Function in Sprague-Dawley Rats.

Manganese (Mn) is an essential nutrient especially during development, but Mn overexposure (MnOE) produces long-term cognitive deficits. Evidence of long-term changes in dopamine in the neostriatum was found in rats from developmental MnOE previously. To examine the relationship between MnOE and dopamine, we tested whether the effects of developmental MnOE would be exaggerated by dopamine reductions induced by 6-hydroxydopamine (6-OHDA) neostriatal infusion when the rats were adults. The experiment consisted of four groups of females and males: Vehicle/Sham, MnOE/Sham, Vehicle/6-OHDA, and MnOE/6-OHDA. Both MnOE/Sham and Vehicle/6-OHDA groups displayed egocentric and allocentric memory deficits, whereas MnOE+6-OHDA had additive effects on spatial memory in the Morris water maze and egocentric learning in the Cincinnati water maze. 6-OHDA reduced dopamine in the neostriatum and nucleus accumbens, reduced norepinephrine in the hippocampus, reduced TH+ cells and TrkB and TH expression in the substantia nigra pars compacta (SNpc), but increased TrkB in the neostriatum. MnOE alone had no effect on monoamines or TrkB in the neostriatum or hippocampus but reduced BDNF in the hippocampus. A number of sex differences were noted; however, only a few significant interactions were found for MnOE and/or 6-OHDA exposure. These data further implicate dopamine and BDNF in the cognitive deficits arising from developmental MnOE.

Prenatal exposure to PCBs in Cyp1a2 knock-out mice interferes with F1 fertility, impairs long-term potentiation, reduces acoustic startle and impairs conditioned freezing contextual memory with minimal transgenerational effects.

Polychlorinated biphenyls (PCBs) are toxic environmental pollutants. Humans are exposed to PCB mixtures via contaminated food or water. PCB exposure causes adverse effects in adults and after exposure in utero. PCB toxicity depends on the congener mixture and CYP1A2 gene activity. For coplanar PCBs, toxicity depends on ligand affinity for the aryl hydrocarbon receptor (AHR). Previously, we found that perinatal exposure of mice to a three-coplanar/five-noncoplanar PCB mixture induced deficits in novel object recognition and trial failures in the Morris water maze in Cyp1a2-/- ::Ahrb1 C57BL6/J mice compared with wild-type mice (Ahrb1  = high AHR affinity). Here we exposed gravid Cyp1a2-/- ::Ahrb1 mice to a PCB mixture on embryonic day 10.5 by gavage and examined the F1 and F3 offspring (not F2 ). PCB-exposed F1 mice exhibited increased open-field central time, reduced acoustic startle, greater conditioned contextual freezing and reduced CA1 hippocampal long-term potentiation with no change in spatial learning or memory. F1 mice also had inhibited growth, decreased heart rate and cardiac output, and impaired fertility. F3 mice showed few effects. Gene expression changes were primarily in F1 PCB males compared with wild-type males. There were minimal RNA and DNA methylation changes in the hippocampus from F1 to F3 with no clear relevance to the functional effects. F0 PCB exposure during a period of rapid DNA de-/remethylation in a susceptible genotype produced clear F1 effects with little evidence of transgenerational effects in the F3 generation. While PCBs show clear developmental neurotoxicity, their effects do not persist across generations for effects assessed herein.

Differential effects of perinatal exposure to antidepressants on learning and memory, acoustic startle, anxiety, and open-field activity in Sprague-Dawley rats.

Most antidepressants inhibit monoamine reuptake. Selective serotonin (5-HT) reuptake inhibitors (SSRIs) act on the 5-HT transporter (SERT) whereas norepinephrine-dopamine reuptake inhibitors (NDRIs) act on the norepinephrine and dopamine transporters. Epidemiological reports link SSRI use during pregnancy to an increased prevalence of autism spectrum disorder (ASD). We previously showed that perinatal exposure to the SSRI citalopram (CIT) results in rodent offspring that exhibit a number of behaviors consistent with an ASD-like phenotype. The present study examined the effect of perinatal exposure to CIT (at a lower dose), another SSRI, fluoxetine (FLX), and an NDRI, bupropion (BUP). Gravid Sprague-Dawley rats were subcutaneously injected twice per day (6h apart) with 5mg/kg CIT, 5mg/kg FLX, 15mg/kg BUP, or saline (SAL) from embryonic day (E) 6-21, and directly to the pups from postnatal day (P) 1-20. As adults, one male/female from each litter was given one of a series of tests. Both SSRI-exposed groups showed spatial learning deficits in Morris and radial water mazes, increased marble burying, increased acoustic startle, hypoactivity, and attenuated activity to the stimulating effect of the NMDA-R antagonist MK-801. The BUP-exposed group showed a reduction in elevated zero-maze quadrant entries and increased stimulated open-field activity following (+)-amphetamine challenge. These results reinforce concern about the use of antidepressants during pregnancy and highlight how the two classes of drugs produce different constellations of effects with more effects associated with the SSRIs. Further investigation into how antidepressants alter brain development leading to enduring adverse neurobehavioral effects is warranted.

Phosphodiesterase-1b (Pde1b) knockout mice are resistant to forced swim and tail suspension induced immobility and show upregulation of Pde10a.

RATIONALE: Major depressive disorder is a leading cause of suicide and disability. Despite this, current antidepressants provide insufficient efficacy in more than 60% of patients. Most current antidepressants are presynaptic reuptake inhibitors; postsynaptic signal regulation has not received as much attention as potential treatment targets. OBJECTIVES: We examined the effects of disruption of the postsynaptic cyclic nucleotide hydrolyzing enzyme, phosphodiesterase (PDE) 1b, on depressive-like behavior and the effects on PDE1B protein in wild-type (WT) mice following stress. METHODS: Littermate knockout (KO) and WT mice were tested in locomotor activity, tail suspension (TST), and forced swim tests (FST). FST was also used to compare the effects of two antidepressants, fluoxetine and bupropion, in KO versus WT mice. Messenger RNA (mRNA) expression changes were also determined. WT mice underwent acute or chronic stress and markers of stress and PDE1B expression were examined. RESULTS: Pde1b KO mice exhibited decreased TST and FST immobility. When treated with antidepressants, both WT and KO mice showed decreased FST immobility and the effect was additive in KO mice. Mice lacking Pde1b had increased striatal Pde10a mRNA expression. In WT mice, acute and chronic stress upregulated PDE1B expression while PDE10A expression was downregulated after chronic but not acute stress. CONCLUSIONS: PDE1B is a potential therapeutic target for depression treatment because of the antidepressant-like phenotype seen in Pde1b KO mice.

Perinatal exposure to the selective serotonin reuptake inhibitor citalopram alters spatial learning and memory, anxiety, depression, and startle in Sprague-Dawley rats.

Selective serotonin reuptake inhibitors (SSRIs) block the serotonin (5-HT) reuptake transporter (SERT) and increase synaptic 5-HT. 5-HT is also important in brain development; hence when SSRIs are taken during pregnancy there exists the potential for these drugs to affect CNS ontogeny. Prenatal SSRI exposure has been associated with an increased prevalence of autism spectrum disorder (ASD), and peripheral 5-HT is elevated in some ASD patients. Perinatal SSRI exposure in rodents has been associated with increased depression and anxiety-like behavior, decreased sociability, and impaired learning in the offspring, behaviors often seen in ASD. The present study investigated whether perinatal exposure to citalopram causes persistent neurobehavioral effects. Gravid Sprague-Dawley rats were assigned to two groups and subcutaneously injected twice per day with citalopram (10mg/kg; Cit) or saline (Sal) 6h apart on embryonic day (E)6-21, and then drug was given directly to the pups after delivery from postnatal day (P)1-20. Starting on P60, one male/female from each litter was tested in the Cincinnati water maze (CWM) and open-field before and after MK-801. A second pair from each litter was tested in the Morris water maze (MWM) and open-field before and after (+)-amphetamine. A third pair was tested as follows: elevated zero-maze, open-field, marble burying, prepulse inhibition of acoustic startle, social preference, and forced swim. Cit-exposed rats were impaired in the MWM during acquisition and probe, but not during reversal, shift, or cued trials. Cit-exposed rats also showed increased marble burying, decreased time in the center of the open-field, decreased latency to immobility in forced swim, and increased acoustic startle across prepulse intensities with no effects on CWM. The results are consistent with citalopram inducing several ASD-like effects. The findings add to concerns about use of SSRIs during pregnancy. Further research on different classes of antidepressants, dose-effect relationships, timing of exposure periods, and mechanisms for these effects are needed. It is also important to balance the effects described here against the effects of the disorders for which the drugs are given.

Developmental manganese exposure in combination with developmental stress and iron deficiency: Effects on behavior and monoamines.

Manganese (Mn) is an essential element but neurotoxic at higher exposures, however, Mn exposure seldom occurs in isolation. It often co-occurs in populations with inadequate dietary iron (Fe) and limited resources that result in stress. Subclinical FeD affects up to 15% of U.S. children and exacerbates Mn toxicity by increasing Mn bioavailability. Therefore, we investigated Mn overexposure (MnOE) in rats in combination with Fe deficiency (FeD) and developmental stress, for which we used barren cage rearing. For barren cage rearing (BAR), rats were housed in cages with a wire grid floor or standard bedding material (STD) from embryonic day (E)7 through postnatal day (P)28. For FeD, dams were fed a 90% Fe-deficient NIH-07 diet from E15 through P28. Within each litter, different offspring were treated with 100mg/kg Mn (MnOE) or vehicle (VEH) by gavage every other day from P4-28. Behavior was assessed at two ages and consisted of: open-field, anxiety tests, acoustic startle response (ASR) with prepulse inhibition (PPI), sociability, sucrose preference, tapered beam crossing, and the Porsolt's forced swim test. MnOE had main effects of decreasing activity, ASR, social preference, and social novelty. BAR and FeD transiently modified MnOE effects. BAR groups weighed less and showed decreased anxiety in the elevated zero maze, had increased ASR and decreased PPI, and exhibited reduced sucrose preference compared with the STD groups. FeD animals also weighed less and had increased slips on the tapered beam. Most of the monoamine effects were dopaminergic and occurred in the MnOE groups. The results showed that Mn is a pervasive developmental neurotoxin, the effects of which are modulated by FeD and/or BAR cage rearing.

Chronic social defeat, but not restraint stress, alters bladder function in mice.

BACKGROUND: Voiding disorders in humans, particularly in children are associated with increased incidence of behavioral issues as well as past history of childhood abuse. We hypothesized that creating stress in mice, utilizing either a chronic social defeat model (SD) or restraint stress in shallow water model (RSSW) would engender changes in bladder function, morphology, and behavior, thereby enabling us to study the resultant voiding dysfunction. METHODS: For SD stress (14 days), C57BL/6 male mice were exposed daily to a larger aggressive CD-1 male for 10 min, followed by sensory exposure in a barrier cage for 24h. Control mice were similarly housed with no exposure. For RSSW (21 days), C57BL/6 mice were put in a perforated conical tube with feet immersed in water daily for 4h, then returned to single housing cages. Control mice were also in single housing. After the stress period, voiding patterns were obtained on filter paper, followed by behavioral tests. At necropsy, blood was taken for corticosterone analysis, and bladder and body weights measured. Bladder cryosections were stained with hematoxylin and eosin (H&E) for morphological assessment. Sequential sections were immunostained with antibodies to Ki-67 as a proliferation marker, CD31 (endothelial cell marker), and uroplakin-II. ImageJ software was used to measure bladder wall thickness on blinded H&E photomicrographs as well as quantitate CD31 staining. Both Ki-67-positive and -negative nuclei were counted with Imaris software to obtain a proliferation index. RESULTS: Only SD mice had a single large void pattern. Bladder-to-body weight ratios increased in SD mice (p≤0.02) but not in RSSW mice. Plasma corticosterone levels were elevated in all stressed mice. SD mice exhibited lower levels of locomotor activity compared with controls; RSSW mice were hyperactive. In SD mice, bladder wall thickness was increased (p ≤ 0.003) but no change was seen in Ki-67 proliferation index, consistent with hypertrophy. No difference with control mice was seen in vascularity as visualized by CD31 staining. Uniform uroplakin-II staining lined the urothelium of both SD and control mice. CONCLUSIONS: Mice exposed to repeated SD (14 days) respond with altered voiding indicative of urine retention, and exhibit bladder wall changes consistent with hypertrophy while the urothelial barrier is maintained. These changes were not observed with repeated RSSW. SD, in contrast to RSSW, provides a model of psychological stress to further study the interplay of behavior and bladder dysfunction, enabling an improved understanding of voiding dysfunction, and the ability to create innovative and more effective management pathways for children who present with voiding dysfunction.