Persistent ∆FosB expression limits recurrent seizure activity and provides neuroprotection in the dentate gyrus of APP mice.

Recurrent seizures lead to accumulation of the activity-dependent transcription factor ∆FosB in hippocampal dentate granule cells in both mouse models of epilepsy and mouse models of Alzheimer's disease (AD), which is also associated with increased incidence of seizures. In patients with AD and related mouse models, the degree of ∆FosB accumulation corresponds with increasing severity of cognitive deficits. We previously found that ∆FosB impairs spatial memory in mice by epigenetically regulating expression of target genes such as calbindin that are involved in synaptic plasticity. However, the suppression of calbindin in conditions of neuronal hyperexcitability has been demonstrated to provide neuroprotection to dentate granule cells, indicating that ∆FosB may act over long timescales to coordinate neuroprotective pathways. To test this hypothesis, we used viral-mediated expression of ∆JunD to interfere with ∆FosB signaling over the course of several months in transgenic mice expressing mutant human amyloid precursor protein (APP), which exhibit spontaneous seizures and develop AD-related neuropathology and cognitive deficits. Our results demonstrate that persistent ∆FosB activity acts through discrete modes of hippocampal target gene regulation to modulate neuronal excitability, limit recurrent seizure activity, and provide neuroprotection to hippocampal dentate granule cells in APP mice.

A Gad2 specific Slc6a8 deletion recapitulates the contextual and cued freezing deficits seen in Slc6a8-/y mice.

The creatine (Cr)-phosphocreatine shuttle is essential for ATP homeostasis. In humans, the absence of brain Cr causes significant intellectual disability, epilepsy, and language delay. Mutations of the creatine transporter (SLC6A8) are the most common cause of Cr deficiency. In rodents, Slc6a8 deletion causes deficits in spatial learning, novel object recognition (NOR), as well as in contextual and cued freezing. The mechanisms that underlie these cognitive deficits are not known. Due to the heterogeneous nature of the brain, it is important to determine which systems are affected by a loss of Cr. In this study, we generated mice lacking Slc6a8 in GABAergic neurons by crossing Slc6a8FL mice with Gad2-Cre mice. These Gad2-specific Slc6a8 knockout (cKO) mice, along with the ubiquitous Slc6a8 KO (Slc6a8-/y), Gad2-Cre+, and wild-type (WT) mice were tested in the Morris water maze, NOR, conditioned freezing, and the radial water maze. Similar to the Slc6a8-/y mice, cKO mice had reduced contextual and cued freezing compared with WT mice. The cKO mice had a mild spatial learning deficit during the reversal phase of the MWM, however they were not as pronounced as in Slc6a8-/y mice. In NOR, the Gad2-Cre mice spent less time with the novel object, similar to the reduced novel time in the cKO mice. There were no changes in radial water maze performance. Slc6a8 deletion in GABAergic neurons is sufficient to recapitulate the conditioned freezing deficits seen in Slc6a8-/y mice.

Interleukin-10-producing monocytes contribute to sex differences in pain resolution in mice and humans.

Pain is closely associated with the immune system, which exhibits sexual dimorphism. For these reasons, neuro-immune interactions are suggested to drive sex differences in pain pathophysiology. However, our understanding of peripheral neuro-immune interactions on sex differences in pain resolution remains limited. Here, we have shown, in both a mouse model of inflammatory pain and in humans following traumatic pain, that males had higher levels of interleukin (IL)-10 than females, which were correlated with faster pain resolution. Following injury, we identified monocytes (CD11b+ Ly6C+ Ly6G-F4/80 mid ) as the primary source of IL-10, with IL-10-producing monocytes being more abundant in males than females. In a mouse model, neutralizing IL-10 signaling through antibodies, genetically ablating IL-10R1 in sensory neurons, or depleting monocytes with clodronate all impaired the resolution of pain hypersensitivity in both sexes. Furthermore, manipulating androgen levels in mice reversed the sexual dimorphism of pain resolution and the levels of IL-10-producing monocytes. These results highlight a novel role for androgen-driven peripheral IL-10-producing monocytes in the sexual dimorphism of pain resolution. These findings add to the growing concept that immune cells play a critical role in resolving pain and preventing the transition into chronic pain.

Developmental deltamethrin: Sex-specific hippocampal effects in Sprague Dawley rats.

Pyrethroid pesticides are widely used and can cause long-term effects after early exposure. Epidemiological and animal studies reveal associations between pyrethroid exposure and altered cognition following prenatal and/or neonatal exposure. However, little is known about the cellular effects of such exposure. Sprague Dawley rats were gavaged with 0 or 1.0 mg/kg deltamethrin (DLM), a Type II pyrethroid, in corn oil (dose volume 5 mL/kg) once per day from postnatal day (P) 3-20 and assessed shortly after dosing ended or as adults. No effects of DLM exposure were found on striatal dopaminergic markers, nor on AMPA receptor subunits or on NMDA-NR1. However, DLM increased NMDA-NR2A and decreased NMDA-NR2B levels in the hippocampus, in males but not females. Additionally, adult hippocampal CA1 long-term potentiation was increased in DLM-treated males but not females. Potassium stimulated extracellular glutamate release in the hippocampus was not affected using in vivo microdialysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed increased apoptotic cells in the dentate gyrus of male rats, in the absence of changes in cleaved caspase-3 at P21. Proinflammatory cytokines interferon gamma trended up in striatum, interleukin-1ß trended down in nucleus accumbens, IL-13 trended up in hippocampus, and keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO or CXCL1) was significantly increased in the hippocampus in male DLM-treated rats on P20. The data point to the developing hippocampus as a susceptible region to DLM-induced adverse effects.

Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model.

Recent studies suggest that ultra-high dose rates of proton radiation (>40 Gy/s; FLASH) confer less toxicity to exposed healthy tissue and reduce cognitive decline compared with conventional radiation dose rates (~1 Gy/s), but further preclinical data are required to demonstrate this sparing effect. In this study, postnatal day 11 (P11) rats were treated with whole brain irradiation with protons at a total dose of 0, 5, or 8 Gy, comparing a conventional dose rate of 1 Gy/s vs. a FLASH dose rate of 100 Gy/s. Beginning on P64, rats were tested for locomotor activity, acoustic and tactile startle responses (ASR, TSR) with or without prepulses, novel object recognition (NOR; 4-object version), striatal dependent egocentric learning ([configuration A] Cincinnati water maze (CWM-A)), prefrontal dependent working memory (radial water maze (RWM)), hippocampal dependent spatial learning (Morris water maze (MWM)), amygdala dependent conditioned freezing, and the mirror image CWM [configuration B (CWM-B)]. All groups had deficits in the CWM-A procedure. Weight reductions, decreased center ambulation in the open-field, increased latency on day-1 of RWM, and deficits in CWM-B were observed in all irradiated groups, except the 5 Gy FLASH group. ASR and TSR were reduced in the 8 Gy FLASH group and day-2 latencies in the RWM were increased in the FLASH groups compared with controls. There were no effects on prepulse trials of ASR or TSR, NOR, MWM, or conditioned freezing. The results suggest striatal and prefrontal cortex are sensitive regions at P11 to proton irradiation, with reduced toxicity from FLASH at 5 Gy.

Latrophilin-3 heterozygous versus homozygous mutations in Sprague Dawley rats: Effects on egocentric and allocentric memory and locomotor activity.

Latrophilin-3 (LPHN3) is a brain specific G-protein coupled receptor associated with increased risk of attention deficit hyperactivity disorder (ADHD) and cognitive deficits. CRISPR/Cas9 was used to generate a constitutive knockout (KO) rat of Lphn3 by deleting exon 3, based on human data that LPHN3 variants are associated with some cases of ADHD. Lphn3 KO rats are hyperactive with an attenuated response to ADHD medication and have cognitive deficits. Here, we tested KO, heterozygous (HET), and wildtype (WT) rats to determine if there was a gene-dosage effect. We tested the rats in home-cage activity starting at postnatal day (P)35 and P50, followed by tests of egocentric learning (Cincinnati water maze [CWM]), spatial learning (Morris water maze [MWM]), working memory (radial water maze [RWM]), incidental learning (novel object recognition [NOR]), acoustic startle response (ASR) habituation, tactile startle response (TSR) habituation, prepulse modification of acoustic startle, shuttle-box passive avoidance, conditioned freezing, and a mirror image version of the CWM. KO and HET rats were hyperactive. KO and HET rats had egocentric (CWM) and spatial deficits (MWM), increased startle response, and KO rats showed less conditioned freezing on contextual and cued memory; there were no effects on working memory (RWM) or passive avoidance. The selective gene-dosage effect in Lphn3 HET rats indicates that Lphn3 exhibits dominate expression on functions where it is most abundantly expressed (striatum, hippocampus) but not on behaviors mediated by regions of low expression. The data add further evidence to the impact of this synaptic protein on brain function and behavior.

Calmodulin acetylation: A modification to remember.

The formation of new memories appears to require alterations in the shape and strength of synapses within the hippocampus, yet our knowledge of the molecular mechanisms underlying these changes remains incomplete. Zhang and colleagues provide new understanding of memory formation by uncovering the lysine acetyltransferase SRC3 as the key driver of the novel posttranslational modification of calmodulin (CaM) acetylation, which regulates CaM's activity and subsequent activation of CaMKII. This new pathway is demonstrated to be both necessary and sufficient for CA3→CA1 synapse long-term potentiation (LTP) and fear memory formation, and this approach may act as a blueprint for future investigation of the role of acetylation of other proteins in neuronal functions.

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.

Effects of Permethrin or Deltamethrin Exposure in Adult Sprague Dawley Rats on Acoustic and Light Prepulse Inhibition of Acoustic or Tactile Startle.

The effects of permethrin (PRM) and deltamethrin (DLM) on acoustic or light prepulse inhibition of the acoustic startle response (ASR) and tactile startle response (TSR) were studied in adult male Sprague Dawley rats. Preliminary studies were conducted to optimize the parameters of light and acoustic prepulse inhibition of ASR and TSR. Once these parameters were set, a new group of rats was administered PRM (0 or 90 mg/kg) or DLM (0 or 25 mg/kg) by gavage in 5 mL/kg corn oil. ASR and TSR were assessed using acoustic or light prepulses 6, 8, and 12 h after PRM and 2, 4, and 6 h after DLM exposure. PRM increased ASR 6 h post-treatment with no interaction with acoustic prepulse levels and with no effect on TSR. When light was used as the prepulse, PRM increased ASR and TSR at 6 h with no interaction with prepulse levels. DLM decreased ASR and TSR on trials without prepulses but not on trials with acoustic prepulses. DLM also decreased ASR when light prepulses were present 4 h post-treatment. A final experiment assessed whether the house light in the test cabinet affected ASR and TSR after PRM or DLM exposure. Rats had increased ASR and TSR when house lights were on compared with when they were off, but lighting did not differentially interact with PRM or DLM. Light and acoustic prepulses of ASR and TSR have different effects depending on the test agent and the test parameters.

Whole brain proton irradiation in adult Sprague Dawley rats produces dose dependent and non-dependent cognitive, behavioral, and dopaminergic effects.

Proton radiotherapy causes less off-target effects than X-rays but is not without effect. To reduce adverse effects of proton radiotherapy, a model of cognitive deficits from conventional proton exposure is needed. We developed a model emphasizing multiple cognitive outcomes. Adult male rats (10/group) received a single dose of 0, 11, 14, 17, or 20 Gy irradiation (the 20 Gy group was not used because 50% died). Rats were tested once/week for 5 weeks post-irradiation for activity, coordination, and startle. Cognitive assessment began 6-weeks post-irradiation with novel object recognition (NOR), egocentric learning, allocentric learning, reference memory, and proximal cue learning. Proton exposure had the largest effect on activity and prepulse inhibition of startle 1-week post-irradiation that dissipated each week. 6-weeks post-irradiation, there were no effects on NOR, however proton exposure impaired egocentric (Cincinnati water maze) and allocentric learning and caused reference memory deficits (Morris water maze), but did not affect proximal cue learning or swimming performance. Proton groups also had reduced striatal levels of the dopamine transporter, tyrosine hydroxylase, and the dopamine receptor D1, effects consistent with egocentric learning deficits. This new model will facilitate investigations of different proton dose rates and drugs to ameliorate the cognitive sequelae of proton radiotherapy.

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.

Impairment of cognitive flexibility in type 2 diabetic db/db mice.

Impaired executive function is a major peril for patients with type 2 diabetes, reducing quality of life and ability for diabetes management. Despite the significance of this impairment, few animal models of type 2 diabetes examine domains of executive function such as cognitive flexibility or working memory. Here, we evaluated these executive function domains in db/db mice, an established model of type 2 diabetes, at 10 and 24 weeks of age. The db/db mice showed impaired cognitive flexibility in the Morris water maze reversal phase. However, the db/db mice did not show apparent working memory disturbance in the spatial working memory version of the Morris water maze or in the radial water maze. We also examined axon initial segments (AIS) and nodes of Ranvier, key axonal domains for action potential initiation and propagation. AIS were significantly shortened in medial prefrontal cortex and hippocampus of 26-week-old db/db mice compared with controls, similar to our previous findings in 10-week-old mice. Nodes of Ranvier in corpus callosum, previously shown to be unchanged at 10 weeks, were elongated at 26 weeks, suggesting an important role for this domain in disease progression. Together, the findings help establish db/db mice as a model of impaired cognitive flexibility in type 2 diabetes and advance our understanding of its pathophysiology.

Mouse knockout of guanylyl cyclase C: Recognition memory deficits in the absence of activity changes.

Guanylyl cyclase C (GC-C) is found in brain regions where dopamine is expressed. We characterized a mouse in which GC-C was knocked out (KO) that was reported to be a model of attention deficit hyperactivity disorder (ADHD). We re-examined this model and controlled for litter effects, used 16 to 23 mice per genotype per sex and assessed an array of behavioral and neurochemical outcomes. GC-C KO mice showed no phenotypic differences from wild-type mice on most behavioral tests, or on striatal or hippocampal monoamines, and notably no evidence of an ADHD-like phenotype. KO mice were impaired on novel object recognition, had decreased tactile startle but not acoustic startle, and females had increased latency on cued training trials in the Morris water maze, but not hidden platform spatial learning trials. Open-field activity showed small differences in females but not males. The data indicate that the GC-C KO mouse with proper controls and sample sizes has a moderate cognitive and startle phenotype but has no ADHD-like phenotype.

Deltamethrin Exposure Daily From Postnatal Day 3-20 in Sprague-Dawley Rats Causes Long-term Cognitive and Behavioral Deficits.

Pyrethroids are synthetic insecticides that act acutely on voltage gated sodium channels to prolong channel opening and depolarization. Epidemiological studies find that exposure to pyrethroids are associated with neurological and developmental abnormalities in children. The long-term effects of type II pyrethroids, such as deltamethrin (DLM), on development have received little attention. We exposed Sprague-Dawley rats to DLM by gavage at doses of 0, 0.25, 0.5, and 1.0 mg/kg/day from postnatal day (P) 3-20 in a split-litter design. Following behavioral testing as adults, monoamine levels, release, and mRNA were assessed via high performance liquid chromatography, microdialysis, and qPCR, respectively. Long-term potentiation (LTP) was assessed at P25-35. Developmental DLM exposure resulted in deficits in allocentric and egocentric learning and memory, increased startle reactivity, reduced conditioned contextual freezing, and attenuated MK-801 induced hyperactivity compared with controls. Startle and egocentric learning were preferentially affected in males. Deltamethrin-treated rats exhibited increased CA1 hippocampal LTP, decreased extracellular dopamine release by microdialysis, reduced dopamine D1 receptor mRNA expression in neostriatum, and decreased norepinephrine levels in the hippocampus. The data indicate that neonatal DLM exposure has adverse long-term effects on learning, memory, startle, glutamatergic function, LTP, and norepinephrine.

Developmental manganese, lead, and barren cage exposure have adverse long-term neurocognitive, behavioral and monoamine effects in Sprague-Dawley rats.

Developmental stress, including low socioeconomic status (SES), can induce dysregulation of the hypothalamic-pituitary-adrenal axis and result in long-term changes in stress reactivity. Children in lower SES households experience more stress and are more likely to be exposed to environmental neurotoxins such as lead (Pb) and manganese (Mn) than children in higher SES households. Co-exposure to stress, Pb, and Mn during early development may increase the risk of central nervous system dysfunction compared with unexposed children. To investigate the potential interaction of these factors, Sprague-Dawley rats were bred, and litters born in-house were culled on postnatal day (P)1 to 6 males and 6 females. One male and female within each litter were assigned to one of the following groups: 0 (vehicle), 10 mg/kg Pb, 100 mg/kg Mn, or 10 mg/kg Pb + 100 mg/kg Mn (PbMn), water gavage, and handled only from P4-28 with half the litters reared in cages with standard bedding (29 litters) and half with no bedding (Barren; 27 litters). Mn and PbMn groups had decreased anxiety, reduced acoustic startle, initial open-field hypoactivity, increased activity following (+)-methamphetamine, deficits in egocentric learning in the Cincinnati water maze (CWM), and deficits in latent inhibition conditioning. Pb increased anxiety and reduced open-field activity. Barren-reared rats had decreased anxiety, CWM deficits, increased startle, and initial open-field hyperactivity. Mn, PbMn, Pb Barren-reared groups had impaired Morris water maze performance. Pb altered neostriatal serotonin and norepinephrine, Mn increased hippocampal serotonin in males, Mn + Barren-rearing increased neostriatal serotonin, and Barren-rearing decreased neostriatal dopamine in males. At the doses used here, most effects were in the Mn and PbMn groups. Few interactions between Mn, Pb, and rearing stress were found, indicating that the interaction of these three variables is not as impactful as hypothesized.