Declan N.C. Jones

Use of SHIRPA and discriminant analysis to characterise marked differences in the behavioural phenotype of six inbred mouse strains

Detailed characterisation of six inbred strains of mice commonly used in transgenic and knockout research was carried out using a battery of behavioural tests (SHIRPA) followed by discriminant analysis of the data. In the primary observation screen, DBA/2 mice were relatively irritable and vocalised during handling. C57BL/6 were hyperactive as measured by transfer arousal, arena activity and touch-escape tests. By contrast, C3H were markedly hypoactive, had significantly enhanced grip strength and were also significantly impaired on the visual placing task. In the elevated plus-maze, BALB/c mice showed the highest level of open arm entries and time spent in the open arms, indicating the lowest level of anxiety. There was a clear dissociation of strains on exploratory activity, as measured in the holeboard test and spontaneous locomotor activity (LMA). DBA/2 mice were hyperactive in LMA but demonstrated relatively low levels of holeboard exploration. None of the six strains learnt the water maze spatial learning task particularly well. C57BL/6 and 129/Sv demonstrated most ability and C3H showed no evidence of having acquired the task. The SHIRPA screening battery and discriminant analysis of the data have enabled us to determine the relevant contribution of a number of behavioural measurements to the marked differences in phenotype of mouse strains. These data confirm the importance of carrying out a comprehensive profile in order to accurately characterise the phenotype of gene-targeted and transgenic mice.

GSK189254, a Novel H3 Receptor Antagonist That Binds to Histamine H3 Receptors in Alzheimer's Disease Brain and Improves Cognitive Performance in Preclinical Models

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H3 receptor antagonist with high affinity for human (pKi = 9.59 –9.90) and rat (pKi = 8.51–9.17) H3 receptors. GSK189254 is >10,000-fold selective for human H3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA2 = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC50 = 8.20 versus basal guanosine 5′-O-(3-[35S]thio)triphosphate binding] at the human recombinant H3 receptor. In vitro autoradiography demonstrated specific [3H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H3 binding was detected in medial temporal cortex samples from severe cases of Alzheimers disease, suggesting for the first time that H3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(–)-α-methyl[imidazole-2,5(n)-3H]histamine dihydrochloride ([3H]R-α-methylhistamine) binding (ED50 = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3–3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID50 = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimers disease and other cognitive disorders.

Long-term behavioural, molecular and morphological effects of neonatal NMDA receptor antagonism

Brief N‐methyl‐d‐aspartate (NMDA) receptor blockade in neonatal rats has been reported to increase neuronal apoptosis. We replicated this finding using MK‐801 (0.5 mg/kg) administered twice on postnatal day 7, and then studied the long‐term consequences. In adulthood, treated rats showed reduced volume and neuronal number within the hippocampus, and altered hippocampal NMDA receptor (NR1 subunit) expression. Synaptophysin mRNA was decreased in the thalamus (laterodorsal nucleus). Adult MK‐801‐treated females had prepulse inhibition deficits and increased locomotor activity. The data show that a transient and limited glutamatergic intervention during development can have chronic behavioural, structural and molecular effects. The effects are reminiscent of alterations reported in schizophrenia and, as such, are consistent with hypotheses advocating a role for NMDA receptor hypofunction, and aberrant apoptosis, in the neurodevelopmental pathogenesis of the disorder.

The behavioural effects of corticotropin-releasing factor-related peptides in rats

Abstract The present study determined the behavioural effects of the corticotropin releasing factor (CRF)-related peptides, human/rat CRF (h/rCRF), ovine CRF (oCRF), sauvagine (SAUV), urotensin I (UT) and the recently discovered neuropeptide, rat urocortin (rUCN). All of the peptides dose-dependently increased motor activity in a familiar environment and reduced feeding in hungry rats. There was no apparent relationship between potency/affinity at CRF2 receptors and effects in these two tests. In a comparison of h/rCRF and rUCN upon discrete spontaneous behaviours, both peptides (3.0 μg ICV) increased activity and grooming, induced a fore-paw tremor and reduced the incidence of motionlessness. However, h/rCRF reduced motionlessness to a greater extent and was a more potent inducer of defaecation, weight loss, oral movements and fore-paw tremor than rUCN. In the elevated X maze, both h/rCRF and rUCN (1.0 μg ICV) had anxiogenic-like effects upon behaviour. In contrast, h/rCRF (1.0 μg ICV), but not rUCN (1.0–10 μg ICV) increased the startle response to an acoustic stimulus. In summary, all the CRF-related peptides increased motor activity and reduced feeding in rats in a similar manner and both rUCN and h/rCRF induced anxiogenesis. However, there were some behavioural differences between rUCN and h/rCRF which require further study. Further pharmacological investigation of the role of CRF receptor subtypes requires the use of subtype selective antagonists.

Medial prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse inhibition of acoustic startle

Rearing rats in isolation produces perturbations in behavior and brain neurochemistry suggested to resemble those of schizophrenia. In particular, isolation-reared rats display deficits in prepulse inhibition of acoustic startle that in humans are associated with disorders including schizophrenia and are interpreted as abnormalities in sensorimotor gating. The prefrontal cortex is considered important in the regulation of prepulse inhibition of acoustic startle and postmortem studies suggest that neuropil and total volume, but not total number of neurons, are decreased in this region of the brains of schizophrenic patients. In this study we used design-based stereological techniques to examine the brains of Lister Hooded rats, reared in isolation and which displayed prepulse inhibition of acoustic startle deficits, for changes in morphology compared with the brains of their socially-reared littermates. Pooled data from three batches of animals revealed a significant 7% volume loss of the medial prefrontal cortex of isolation-reared rats whereas neuron number in this region was unchanged. In contrast, volume and total neuron number were unaffected in the rostral caudate putamen. The robust reduction in prefrontal cortical volume observed in isolation-reared rats, in the absence of reductions in total neuron number, suggest that there is a loss of volume of the neuropil. These changes parallel those reported in schizophrenia patients and therefore support the construct validity of this model.

Comparison between intraperitoneal and subcutaneous phencyclidine administration in Sprague–Dawley rats: A locomotor activity and gene induction study

In a putative model of acute phencyclidine (PCP)-induced psychosis we evaluated effects of the drug on locomotor activity (LMA) and immediate early gene (IEG) induction in the rat using two routes of drug administration, intraperitoneal (i.p.) and subcutaneous (s.c.). Adult male rats received saline or PCP (1.0-5.0 mg/kg) either i.p or s.c. and were assessed for LMA for 60 min. At the end of the LMA testing animals were culled and blood and brain samples were collected for PCP concentration analysis. Separate cohorts of animals received 5.0 mg/kg PCP (i.p. or s.c.) and were used to investigate (1) the pharmacokinetics of PCP or (2) induction of IEG (Arc, c-fos, BDNF, junB, Krox-20, sgk-1, NURR1, fra-2, Krox-24, and egr-3) mRNA expression in the prefrontal cortex (PFC). Administration of PCP resulted in locomotor hyperactivity which was more robust and longer-lasting in animals dosed s.c. compared to i.p.-treated-animals. Differences in hyperlocomotion were paralleled by higher concentrations of PCP in the blood and in the brain of s.c.-treated animals compared to i.p.-treated animals. The differences in the concentration of PCP between the two routes of administration were detected 30 min after dosing and persisted for up to 4 h. Administration of PCP via the s.c. route resulted in induction of more IEGs and consistently larger magnitudes of induction than that via the i.p. route. Therefore, we have outlined the dosing conditions to induce rapid and robust effect of acute PCP on behaviour, gene induction, and pharmacokinetic profile, to allow investigation of this as a potential animal model of acute psychosis.

Evaluation of the spontaneously hypertensive rat as a model of attention deficit hyperactivity disorder: acquisition and performance of the DRL-60s test

CD. However, the pattern of responding at DRL-60s suggested poor schedule control for the WKY rats. Therefore, the performance of SHR in the DRL test does not appear to represent a valid model of ADHD. Further, our findings with the WKY rat suggest that this strain is a poor behavioural control for the SHR.

Antipsychotic Treatment Alters Protein Expression Associated with Presynaptic Function and Nervous System Development in Rat Frontal Cortex

Haloperidol and olanzapine are widely used antipsychotic drugs in the treatment of schizophrenia and other psychotic disorders. Despite extensive research efforts within the biopharmaceutical industry and academia, the exact molecular mechanisms of their action remain largely unknown. Since the response of patients to existing medications can be variable and often includes severe side effects, it is critical to increase our knowledge on their mechanism of action to guide clinical usage and new drug development. In this study, we have employed the label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify differentially expressed proteins in rat frontal cortex following subchronic treatment with haloperidol or olanzapine. Subcellular fractionation was performed to increased proteomic coverage and provided insight into the subcellular location involved in the mechanism of drug action. LC-MSE profiling identified 531 and 741 annotated proteins in fractions I (cytoplasmic-) and II (membrane enriched-) in two drug treatments. Fifty-nine of these proteins were altered significantly by haloperidol treatment, 74 by olanzapine and 21 were common to both treatments. Pathway analysis revealed that both drugs altered similar classes of proteins associated with cellular assembly/organization, nervous system development/function (particularly presynaptic function) and neurological disorders, which indicate a common mechanism of action. The top affected canonical signaling pathways differed between the two treatments. The haloperidol data set showed a stronger association with Huntingtons disease signaling, while olanzapine treatment showed stronger effects on glycolysis/gluconeogenesis. This could either relate to a difference in clinical efficacy or side effect profile of the two compounds. The results were consistent with the findings reported previously by targeted studies, demonstrating the validity of this approach. However, we have also identified many novel proteins which have not been found previously to be associated with these drugs. Further study of these proteins could provide new insights into the etiology of the disease or the mechanism of antipsychotic medications.

Characterization of the binding of [125I]‐human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor

GPR10 is a novel G‐protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor‐1 (UHR‐1). Human prolactin‐releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [125I]‐PrRP‐20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [125I]‐PrRP‐20 was saturable, and analysis suggested evidence of both high and low affinity sites, with KD values of 0.026±0.006 and 0.57±0.14 nM respectively, and Bmax values of 3010±400 and 8570±2240 fmol mg protein−1 respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a KD of 0.012 nM. Competition studies revealed that human and rat PrRP‐20 and PrRP‐31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF‐amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP‐20 failed to alter basal or forskolin‐stimulated levels of intracellular cyclic AMP in HEK293‐GPR10 cells, suggesting that GPR10 does not couple via either Gs or Gi. Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP‐20 and PrRP‐31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca2+ stores. These studies indicate that [125I]‐PrRP‐20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.

Cellular uptake and spread of the cell‐permeable peptide penetratin in adult rat brain

Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell‐permeable peptide consisting of a 16‐amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion‐fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 μg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose‐dependent fashion. Doses of 1 μg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 μg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.