Henry J. Olverman

Memory reconsolidation: sensitivity of spatial memory to inhibition of protein synthesis in dorsal hippocampus during encoding and retrieval.

Reconsolidation is a putative neuronal process in which the retrieval of a previously consolidated memory returns it to a labile state that is once again subject to stabilization. This study explored the idea that reconsolidation occurs in spatial memory when animals retrieve memory under circumstances in which new memory encoding is likely to occur. Control studies confirmed that intrahippocampal infusions of anisomycin inhibited protein synthesis locally and that the spatial training protocols we used are subject to overnight protein synthesis-dependent consolidation. We then compared the impact of anisomycin in two conditions: when memory retrieval occurred in a reference memory task after performance had reached asymptote over several days; and after a comparable extent of training of a delayed matching-to-place task in which new memory encoding was required each day. Sensitivity to intrahippocampal anisomycin was observed only in the protocol involving new memory encoding at the time of retrieval.

Increased Expression of the 5-HT Transporter Confers a Low- Anxiety Phenotype Linked to Decreased 5-HT Transmission

A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.

Behavioural analysis of the acute and chronic effects of MDMA treatment in the rat.

Abstract  Rationale: A variety of animal models have shown MDMA (3,4-methylenedioxymethamphetamine) to be a selective 5-HT neurotoxin, though little is known of the long-term behavioural effects of the pathophysiology. The widespread recreational use of MDMA thus raises concerns over the long-term functional sequelae in humans. Objective: This study was designed to explore both the acute- and post-treatment consequences of a 3-day neurotoxic exposure to MDMA in the rat, using a variety of behavioural paradigms. Methods: Following training to pretreatment performance criteria, animals were treated twice daily with ascending doses of MDMA (10, 15, 20 mg/kg) over 3 days. Body temperature, locomotor activity, skilled paw-reaching ability and performance of the delayed non-match to place (DNMTP) procedure was assessed daily during this period and on an intermittent schedule over the following 16 days. Finally, post mortem biochemical analyses of [3H] citalopram binding and monoamine levels were performed. Results: During the MDMA treatment period, an acute 5-HT-like syndrome was observed which showed evidence of tolerance. Once drug treatment ceased the syndrome abated completely. During the post-treatment phase, a selective, delay-dependent, deficit in DNMTP performance developed. Post-mortem analysis confirmed reductions in markers of 5-HT function, in cortex, hippocampus and striatum. Conclusions: These results confirm that acutely MDMA exposure elicits a classical 5-HT syndrome. In the long-term, exposure results in 5-HT neurotoxicity and a lasting cognitive impairment. These results have significant implications for the prediction that use of MDMA in humans could have deleterious long-term neuropsychological/psychiatric consequences.

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists.

1 The pharmacological profile of adenosine A1 receptors in human, guinea‐pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]‐8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]‐DPCPX). 2 The affinity of [3H]‐DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea‐pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea‐pig or human cortical membranes. 3 The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5′‐N‐ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5–26.2 fold higher in rat cortical membranes than in human and guinea‐pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 μM) reduced 2‐chloro‐N6‐cyclopentyladenosine (CCPA) affinity 7–13.9 fold, whereas the affinity of DPCPX was unaffected. 4 The affinity of six xanthine‐based adenosine receptor antagonists was 2.2–15.9 fold higher in rat cortical membranes compared with human or guinea‐pig membranes. The rank order of potency was species‐independent. In contrast, three pyrazolopyridine derivatives, (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐2‐piperidine ethanol (FK453), (R)‐1‐[(E)‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl) acryloyl]‐piperidin‐2‐yl acetic acid (FK352) and 6‐oxo‐3‐(2‐phenylpyrazolo[1,5‐a]pyridin‐3‐yl)‐1(6H)‐pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea‐pig, rat and mouse brain membranes. pKi values (M) for [3H]‐DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5 Drug affinity for adenosine A2A receptors was determined in a [3H]‐2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([3H]‐CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]‐DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]‐DPCPX binding sites in human, rat, mouse and guinea‐pig brain membranes.

Functional consequences of perinatal exposure to3,4-methylenedioxymethamphetamine in rat brain

In this study we have examined methylenedioxymethamphetamine (MDMA)‐induced toxicity in perinatal rat brain, related this to normal development of serotonin transporter sites (SERT), and determined whether early exposure to MDMA subsequently alters cerebral function in adults. Perinatal development of SERT was visualized and quantified using [3H]‐paroxetine binding autoradiography in embryonic and neonatal rat brain from embryonic day 15 (E15) to postnatal day 30 (P30). Cerebral glucose utilization (lCMRglu) was measured by 2‐deoxyglucose autoradiography in adult rats. [3H]‐Paroxetine binding was observed in forebrain from E18. From birth (P0), binding was organized into neocortical columns (75% higher at P10 than in adult) which declined toward adult levels between P20 and P25. MDMA treatment (20 mg kg−1 s.c. twice daily for four days) commencing at developmental stages from E15 (treatment given to dams) to P20, had no effect upon [3H]‐paroxetine binding measured at P40. Treatments started on P25 or later resulted in significant decreases in [3H]‐paroxetine binding (46%). This was coincident with the development of adult patterns of binding in forebrain. Despite the lack of MDMA‐induced neurotoxicity, rats treated in utero (E15) showed increased lCMRglu in locus coeruleus (+37%), and in areas receiving ascending noradrenergic innervation, such as anterior thalamus (+44%) and septal nucleus (+24%). These studies confirm that the susceptibility of serotonergic terminals to the neurotoxic properties of MDMA is absent in the immediate perinatal period, but also suggests that in utero MDMA exposure produces significant long‐term effects on cerebral function by a mechanism as yet unknown.

Molecular target size analyses of the NMDA-receptor complex in rat cortex

The molecular weights of different subunits of the NMDA-receptor complex were determined by high-energy radiation inactivation analyses of the binding of [3H]L-glutamate, [3H](3-(+/-)-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) and [3H]glycine to rat cortical membranes. The molecular target sizes of [3H]L-glutamate binding (the recognition site), [3H]TCP binding (the ionophore) and [3H]glycine (a modulatory unit) were similar: 121,000, 118,000 and 115,000 Da, respectively. These results suggest that the three subunits are on the same protein. The molecular weight of [3H]CPP binding was 209,000 Da. This suggests that in order to bind [3H]CPP (a competitive antagonist) with high affinity an additional macromolecule may be associated to the agonist site.

[3H]p-aminoclonidine and [3H]idazoxan label different populations of imidazoline sites on rat kidney

In the presence of RS-15385-197 to preclude binding to alpha 2-adrenoceptors, [3H]p-aminoclonidine labelled a low affinity high capacity site, (Kd = 127.6 +/- 19.7 nM, Bmax 978 +/- 172 fmol/mg protein) whereas [3H]idazoxan labelled a high affinity low capacity site (Kd = 1.66 +/- 0.28 nM, Bmax 45.3 +/- 11.4 fmol/mg protein). Clonidine and p-aminoclonidine showed moderate affinity for the site labelled by [3H]p-aminoclonidine, but low affinity for the site labelled by [3H]idazoxan, whereas idazoxan showed high affinity for [3H]idazoxan and low affinity for [3H]p-aminoclonidine binding. Naphazoline inhibited [3H]idazoxan in a biphasic manner suggesting that [3H]idazoxan may label an heterogeneous population of imidazoline sites. GTP inhibited [3H]idazoxan but not [3H]p-aminoclonidine binding. These results suggest that [3H]idazoxan labelled imidazoline I2 binding sites, whereas [3H]p-aminoclonidine labelled a novel subtype which showed marked differences to the imidazoline I1 binding site reported in bovine and human brainstem.

Novel analysis for improved validity in semi-quantitative 2-deoxyglucose autoradiographic imaging

The original [(14)C]-2-deoxyglucose autoradiographic imaging technique allows for the quantitative determination of local cerebral glucose utilisation (LCMRglu) [Sokoloff L, Reivich, M, Kennedy C, Desrosiers M, Patlak C, Pettigrew K, et al. The 2-deoxyglucose-C-14 method for measurement of local cerebral glucose utilisation-theory, procedure and normal values in conscious and anestherized albino rats. J Neurochem 1977;28:897-916]. The range of applications to which the quantitative method can be readily applied is limited, however, by the requirement for the intermittent measurement of arterial radiotracer and glucose concentrations throughout the experiment, via intravascular cannulation. Some studies have applied a modified, semi-quantitative approach to estimate LCMRglu while circumventing the requirement for intravascular cannulation [Kelly S, Bieneman A, Uney J, McCulloch J. Cerebral glucose utilization in transgenic mice over-expressing heat shock protein 70 is altered by dizocilpine. Eur J Neurosci 2002;15(6):945-52; Jordan GR, McCulloch J, Shahid M, Hill DR, Henry B, Horsburgh K. Regionally selective and dose-dependent effects of the ampakines Org 26576 and Org 24448 on local cerebral glucose utilisation in the mouse as assessed by C-14-2-deoxyglucose autoradiography. Neuropharmacology 2005;49(2):254-64]. In this method only a terminal blood sample is collected for the determination of plasma [(14)C] and [glucose] and the rate of LCMRglu in each brain region of interest (RoI) is estimated by comparing the [(14)C] concentration in each region relative to a selected control region, which is proposed to demonstrate metabolic stability between the experimental groups. Here we show that the semi-quantitative method has reduced validity in the measurement of LCMRglu as compared to the quantitative method and that the validity of this technique is further compromised by the inability of the methods applied within the analysis to appropriately determine metabolic stability in the selected standard region. To address these issues we have developed a novel form of analysis that provides an index of LCMRglu (iLCMRglu) for application when using the semi-quantitative approach. Provided that the methodological constraints inherent in 2-deoxyglucose autoradiography (e.g. normoglycaemia) are met this analytical technique both increases the validity of LCMRglu estimation by the semi-quantitative method and also allows for its broader experimental application.

Chronic Activation of Corticotropin-Releasing Factor Type 2 Receptors Reveals a Key Role for 5-HT1A Receptor Responsiveness in Mediating Behavioral and Serotonergic Responses to Stressful Challenge

Background The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation. Methods Transgenic mice overexpressing the highly specific CRFR2 ligand urocortin 3 (UCN3OE) were analyzed for stress-related behaviors and hypothalamic-pituitary-adrenal axis responses. Responses to 5-HT receptor agonist challenge were assessed by local cerebral glucose utilization, while 5-HT and 5-hydroxyindoleacetic acid content were quantified in limbic brain regions. Results Mice overexpressing urocortin 3 exhibited increased stress-related behaviors under basal conditions and impaired retention of spatial memory compared with control mice. Following acute stress, unlike control mice, they exhibited no further increase in these stress-related behaviors and showed an attenuated adrenocorticotropic hormone response. 5-HT and 5-hydroxyindoleacetic acid content of limbic nuclei were differentially regulated by stress in UCN3OE mice as compared with control mice. Responses to 5-HT type 1A receptor challenge were significantly and specifically reduced in UCN3OE mice. The distribution pattern of local cerebral glucose utilization and 5-HT type 1A receptor messenger RNA expression levels suggested this effect was mediated in the raphé nuclei. Conclusions Chronic activation of CRFR2 promotes an anxiety-like state, yet with attenuated behavioral and hypothalamic-pituitary-adrenal axis responses to stress. This is reminiscent of stress-related atypical psychiatric syndromes such as posttraumatic stress disorder, chronic fatigue, and chronic pain states. This new understanding indicates CRFR2 antagonism as a potential novel therapeutic target for such disorders.

Effects of the specific serotonin reuptake inhibitor, citalopram, upon local cerebral blood flow and glucose utilisation in the rat

The effects of the potent selective 5-HT reuptake blocking agent, citalopram (10 mg/kg, i.v.), on local cerebral blood flow (lCBF) and local cerebral metabolic rate of glucose (lCMRglu) were measured using [14C]iodoantipyrine (IAP) and [14C]2-deoxyglucose (2-DG) autoradiography, respectively. Significant decreases in lCBF were observed in nine of the 27 brain areas analysed, with significant decreases in lCMRglu observed in 17 areas. While decreases in blood flow were observed, it cannot be concluded that these were in fact the result of a direct action of 5-HT upon serotonergic receptors in cerebrovascular smooth muscle, since the dynamic relationship between flow and metabolism remains largely intact. The reductions in lCBF may be explained entirely by the secondary effects of depressed cerebral metabolic demand induced by citalopram which would, once again, question the role of specifically perivascular serotonergic nerve activity in the tonic control of cerebral blood flow.