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A team of NHS clinicians explain how mobile cancer care units, provided by the charity Hope For Tomorrow, have helped them to deliver less disruptive cancer treatment and continue to care for patients during COVID-19.

Kristina Medlinskiene,1,2 Susan Richardson,3 Beth Fylan,1,4,5 Katherine Stirling,2 Marcus Rattray,1,4 Duncan Petty1 1Medicine Optimisation Research Group, School of Pharmacy and Medical Sciences, University of Bradford, Bradford, UK; 2Medicine Management and Pharmacy Services, Leeds Teaching Hospitals NHS Trust, Leeds, UK; 3Department of Management, Huddersfield Business School, University of Huddersfield, Huddersfield, HD1 3DH, UK; 4Wolfson Centre for Applied Health Research, Bradford, BD9 6RJ, UK; 5Bradford Institute for Health Research, NIHR Patient Safety Translational Research Centre, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, BD9 6RJ, UKCorrespondence: Kristina Medlinskiene Email kristina.medlinskiene@nhs.netIntroduction: Oral anticoagulant therapy choices for patients with atrial fibrillation (AF) expanded in the last decade with the introduction of direct oral anticoagulants (DOAC). However, the implementation of DOACs was slow and varied across different health economies in England. There is limited evidence on the patient role in the uptake of new medicines, including DOACs, apart from considering their demographic and clinical characteristics. Hence, this study aimed to address the gap by exploring the view of patients with AF on factors affecting DOAC use.Methods: A qualitative study using semi-structured interviews was conducted in three health economies in the North of England. Adult patients (> 18 years) diagnosed with non-valvular AF, prescribed an oral anticoagulant (vitamin K antagonist or DOAC), and able to give written consent were recruited. Data were collected between August 2018 and April 2019. Audio recorded interviews were transcribed verbatim and analyzed using the framework method.Results: Four themes with eleven subthemes discussed identified factors affecting the use of DOACs. They were linked to limited healthcare financial and workforce resources, patient involvement in decision-making, patient knowledge about DOACs, safety concerns about oral anticoagulants, and oral anticoagulant therapy impact on patients’ daily lives. Lack of a) opportunities to voice patient preferences and b) information on available therapy options resulted in some patients experiencing difficulties with the prescribed therapy. This was reported to cause negative impact on their daily lives, adherence, and overall satisfaction with the therapy.Conclusion: Greater patient involvement in decision-making could prevent and resolve difficulties encountered by some patients and potentially improve outcomes plus increase the uptake of DOACs.Keywords: uptake, new medicines, shared decision making, direct oral anticoagulants, DOACs, warfarin

Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. Electronic supplementary material The online version of this article (10.1007/s11064-019-02832-2) contains supplementary material, which is available to authorized users.

Introduction Acknowledging patients’ preferences and addressing their concerns is thought to improve patients’ satisfaction with consultations, resulting in improved adherence and health benefits [1]. Adherence to oral anticoagulant (OA) therapy in non-valvular atrial fibrillation (NVAF) is crucial to prevent AF-related strokes. Previous research on anticoagulation in NVAF indicated that decision-making was dominated by prescribers, but patients offered several therapy options reported being involved [2]. Identifying the patient’s perspective on factors affecting their involvement could help to deliver more patient-centred care in the management of AF. Aim To explore patients’ perspectives and experiences concerning factors affecting their involvement in decision-making about OA therapy for stroke prevention in NVAF. Methods Semi-structured face-to-face interviews were conducted with patients in three health economies in the North of England between August 2018 and April 2019. An interview topic guide based on narrative review findings [2] was developed and refined by a Patient and Public Involvement (PPI) group to ensure clarity. Theoretical sampling was used to recruit adults (>18 years) diagnosed with NVAF, prescribed OA (vitamin K antagonist or direct oral anticoagulant (DOAC)), and able to give written consent. Up to 30 potential participants were approached by a member of their direct care team (nurse or pharmacist) in secondary and primary care anticoagulation or arrhythmia clinics. PPI members recruited patients from a local patient support group. Data collection continued until data saturation was achieved. Audio recorded interviews were transcribed verbatim and analysed using the Framework method. Results A total of 21 patient with median (IQR) age of 73 years (65–78 years) were interviewed. Eleven patients took DOACs. The median (IQR) time of taking OA was 3 years (4 weeks-7 years), warfarin 7 years (3–9 years) and DOAC 1 year (4 weeks-4 years). Interviews lasted between 15 and 48 minutes (average 27 minutes). Three themes were identified during analysis (Figure 1). Patients described therapy decisions being made by just the clinician, jointly with the clinician, and in few cases by the patient alone. The lack of involvement resulted from patients perceiving that there was only a choice between accepting and refusing the therapy, and clinician’s role was to make decisions. Limited consultation times, lack of continuous relationship with a clinician and knowledge about OAs options discouraged patients from being involved and some patients reported difficulties with the prescribed therapy. Patients that were involved considered safety of therapy options and impact on daily life. Conclusions Limited consultation time, patient’s perception of clinician’s role, quality of patient-clinician relationship, and patients’ awareness of therapy options were main factors influencing the level of patient involvement in the decision-making. Support from both organisation and clinicians (e.g., longer consultations, availability of different therapy options) is needed to facilitate greater patient involvement, which could prevent difficulties encountered by some patients. The main strength of this study was recruitment of participants from three health economies differing in anticoagulation service provision models, which enabled greater insight into potential barriers. However, the sample was lacking ethnic diversity and could have potential of recall bias. References 1. Shay LA and Lafata JE. (2015). Where is the evidence? A systematic review of shared decision making and patient outcomes. Medical Decision Making. 35 (1): 114–131. 2. Medlinskiene K, Petty D, Richardson S and Stirling K. (2018) Are patients with non-valvular atrial fibrillation involved in decision-making about oral anticoagulants? A literature review. International Journal of Pharmacy Practice. 26 (Suppl. 1): 42–43.

[Image: see text] Despite intense attention from biomedical and chemical researchers, there are few approved treatments for amyotrophic lateral sclerosis (ALS), with only riluzole (Rilutek) and edaravone (Radicava) currently available to patients. Moreover, the mechanistic basis of the activity of these drugs is currently not well-defined, limiting the ability to design new medicines for ALS. This Letter describes the synthesis of triazole-containing riluzole analogues, and their testing in a novel neuroprotective assay. Seven compounds were identified as having neuroprotective activity, with two compounds having similar activity to riluzole.

Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer’s disease. These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease modifying therapies for Alzheimer’s disease, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of Alzheimer’s disease. A small number of the four hundred transporter superfamilies’ are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in Alzheimer’s disease. Here we review the current evidence for six astrocytic transporter subfamilies involved in Alzheimer’s disease, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling Alzheimer’s disease.

Sonic hedgehog (SHH) is a glycoprotein associated with development that is also expressed in the adult CNS and released after brain injury. Since the SHH receptors patched homolog‐1 and Smoothened are highly expressed on astrocytes, we hypothesized that SHH regulates astrocyte function. Primary mouse cortical astrocytes derived from embryonic Swiss mouse cortices, were treated with two chemically distinct agonists of the SHH pathway, which caused astrocytes to elongate and proliferate. These changes are accompanied by decreases in the major astrocyte glutamate transporter‐1 and the astrocyte intermediate filament protein glial fibrillary acidic protein. Multisite electrophysiological recordings revealed that the SHH agonist, smoothened agonist suppressed neuronal firing in astrocyte‐neuron co‐cultures and this was abolished by the astrocyte metabolic inhibitor ethylfluoroacetate, revealing that SHH stimulation of metabolically active astrocytes influences neuronal firing. Using three‐dimensional co‐culture, MAP2 western blotting and immunohistochemistry, we show that SHH‐stimulated astrocytes protect neurons from kainate‐induced cell death. Altogether the results show that SHH regulation of astrocyte function represents an endogenous neuroprotective mechanism.

Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and 3H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 μM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 μM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 μM – 1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29-30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a 'reactive' response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.

The l-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular l-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent l-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Dietary flavonoids, including the citrus flavanone hesperetin, may have stimulatory effects on cytoprotective intracellular signalling pathways. In primary mouse cortical neurone cultures, but not SH-SY5Y human neuroblastoma cells or human primary dermal fibroblasts (Promocells), hesperetin (100–300 nM, 15 min) caused significant increases in the level of ERK1/2 phosphorylation, but did not increase CREB phosphorylation. Administration of hesperetin for 18 h did not alter gene expression driven by the cyclic AMP response element (CRE), assessed using a luciferase reporter system, but 300 nM hesperetin partially reversed staurosporine-induced cell death in primary neurones. Our data show that hesperetin is a neuroprotective compound at concentrations where antioxidant effects are unlikely to predominate. The effects of hesperetin are cell-type dependent and, unlike the flavanol (−)epicatechin, neuroprotection in vitro is not associated with enhanced CREB phosphorylation or CRE-mediated gene expression.

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.

After a single intraperitoneal injection of the irreversible tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg), there was a rapid down-regulation of serotonin (5-HT) transporter mRNA levels in cell bodies. This change was significant at 1 and 2 days after PCPA administration within the ventromedial but not the dorsomedial portion of the dorsal raphe nucleus. Seven days after PCPA treatment, 5-HT transporter mRNA levels were significantly elevated compared with controls in both regions of the dorsal raphe nucleus. PCPA administration produced no change in the [3H]citalopram binding and synaptosomal [3H]5-HT uptake in terminal regions at 2 and 7 days after treatment but significantly reduced both these parameters by approximately 20% in the hippocampus and in cerebral cortex 14 days after PCPA administration. The striatum showed a lower sensitivity to this effect. No significant changes were observed in the levels of [3H]citalopram binding to 5-HT cell bodies in the dorsal raphe nucleus. In the same animals used for 5-HT transporter mRNA level measurements, levels of tryptophan hydroxylase mRNA in neurons of the ventromedial and dorsomedial portions of the dorsal raphe nucleus were increased 2 days after PCPA administration and fell to control levels 7 days after injection in the ventromedial region but not in the dorsomedial portion of the dorsal raphe nucleus, where they remained significantly higher than controls. Altogether, these results show that changes in 5-HT transporter mRNA are not temporally related to changes in 5-HT transporter protein levels. In addition, our results suggest that the 5-HT transporter and tryptophan hydroxylase genes are regulated by different mechanisms. We also provide further evidence that dorsal raphe 5-HT neurons are differentially regulated by drugs, depending on their location.

The selective group-III metabotropic glutamate receptor agonist, L-serine-O-phosphate (L-SOP), when injected bilaterally into the inferior colliculus of the sound sensitive genetically epilepsy-prone (GEP) rats produces a short proconvulsant excitation followed by a long phase of protection against sound-induced seizures lasting for 2-4 days. We have studied this prolonged suppression of audiogenic seizures using pharmacological and molecular biological approaches including semiquantitative RT-PCR and western blotting. The intracerebroventricular injection of the protein synthesis inhibitor cycloheximide (120 microg) 30 min beforehand significantly reduces the proconvulsant seizure activity and the prolonged anticonvulsant effect of intracollicular L-SOP (500 nmol/side). The sensitive semiquantitative RT-PCR revealed a significant up-regulation in mGlu(4) and mGlu(7) mRNA levels in the inferior colliculus at 2 days (maximum suppression of audiogenic seizures) after intracollicular L-SOP injection compared with the non-injected, 2-day post-vehicle treated and 7-day (return to expressing audiogenic seizures) post-drug or vehicle-treated groups. No significant changes were observed in mGlu(6) or mGlu(8) mRNA expression levels in drug-treated compared with control groups. Examination of mGlu(4a) and mGlu(7a) protein levels using western blotting showed a significant increase in mGlu(7a) but no significant change in mGlu(4a) protein levels 2 days after L-SOP treatment compared with the control groups (non-injected and 2-day vehicle-injected group). These results suggest that up-regulation of mGlu(7) receptors is involved in the prolonged anticonvulsant effect of L-SOP against sound-induced seizures in GEP rats. The potential use of mGlu(7) agonists as novel anti-epileptic agents merits investigation.

The 5-HT(1A) receptor has previously been shown to be important in mediating the behavioural effects of nicotine. It is possible that nicotine administration might regulate the levels of 5-HT receptors in limbic and cortical regions, and such regulations may underlie adaptive responses to nicotine in the central nervous system. The effects of acute and chronic systemic (--)-nicotine administration on 5-HT(1A) receptor gene expression were measured by in situ hybridization, in the rat cerebral cortex, dorsal hippocampus and lateral septum. In the cortex, acute nicotine (0.5 mg/kg i.p.) significantly increased the expression of 5-HT(1A) receptor mRNA 2 h and 24 h after injection. Similarly, acute nicotine significantly increased 5-HT(1A) receptor mRNA in the dentate gyrus (DG), CA3 and CA1 regions of the dorsal hippocampus 2 h and 24 h after injection. Acute nicotine was without effect in the lateral septum. Chronic nicotine (0.5 mg/kg i.p; twice daily for 7 days) significantly decreased 5-HT(1A) receptor mRNA in the cortex 2 h after the final injection, but was without effect at 24 h or 72 h. Chronic nicotine caused no changes in 5-HT(1A) mRNA in the lateral septum or dorsal hippocampus. These data demonstrate that nicotine regulates 5-HT(1A) receptor gene expression in the cortex and hippocampus. The rapid regulation of expression of 5-HT(1A) receptor mRNA leads to the hypothesis that nicotine-induced 5-HT release may alter the postsynaptic sensitivity to 5-HT.

Cholinergic receptor agonists nicotine (nicotinic), carbachol (nicotinic/muscarinic) and pilocarpine (muscarinic) were administered into the hippocampus and mRNA levels of neurotrophins and their receptors determined using in situ hybridisation. Drug doses were carefully chosen to avoid the potentially confounding effects of seizure and cell death. Nicotine caused a long-lasting increase in nerve growth factor (NGF) mRNA in all subfields of the hippocampus. The increase was evident from 24 h up to 72 h after drug administration. This increase was dependent on excitatory amino acid neurotransmission as it was blocked by administration of an AMPA or NMDA receptor antagonist. In contrast, carbachol and pilocarpine produced a transient increase in NGF mRNA levels present 4-8 h after drug administration. Pilocarpine caused a transient increase in hippocampal brain-derived neurotrophic factor (BDNF) levels, with carbachol and nicotine showing the same trend. Nicotine and carbachol caused transient decreases in NT-3 mRNA levels in dentate gyrus and CA2 with pilocarpine showing a similar trend. Increases in mRNA encoding full-length trkB were seen 8 h after nicotine, with nicotine also causing elevations in a mRNA encoding a truncated isoform (trkB.T2). TrkC mRNA was not altered by any of the conditions used. The study suggests that muscarinic and nicotinic receptor activation in the hippocampus causes transient changes in all of the neurotrophins, but that NGF levels are selectively up-regulated by nicotinic receptor stimulation. The reciprocal interaction between NGF and ascending cholinergic systems may be a component of the cognitive enhancing effects of nicotine.

The effect of short-term treatment of normal or hypophysectomized rats with biosynthetic growth hormone (GH) was studied in extensor digitorum longus and soleus muscles. In situ hybridization revealed that in normal rats, mRNA for collagen I, collagen III and insulin-like growth factor-I (IGF-I) are expressed by fibroblasts between the muscle fibre areas and that the specificity of this location was not altered by GH administration. Hypophysectomy appeared to cause a decrease in IGF-I and decreased collagen I and III gene expression (P0.001, P0.001, respectively). GH administration seemed to increase IGF-I mRNA levels in all the animals studied. Quantitative image analysis that GH administration to hypophysectomized rats caused an increase in collagen I gene expression after 2 days (P0.05) and an increase in collagen III gene expression after 4 days (P0.05). The results indicate that the fibroblast cells are an important target for the action of GH on skeletal muscle and that the fibroblasts respond to GH by increases in the expression of mRNA for collagen I and collagen III.

The polymerase chain reaction was utilized to determine which of six cloned phospholipase A2 (PLA2) messenger RNAs (mRNAs), encoding four distinct low molecular weight forms of secretory PLA2 (PLA2-IB, PLA2-IIA, PLA2-IIC and PLA2-V), a calcium-dependent high molecular weight PLA2 (PLA2-IV, cPLA2) and a calcium-independent high molecular weight PLA2 (PLA2-VI, iPLA2), were expressed in different regions of rat brain and in a number of peripheral tissues. Pancreatic PLA2-IB mRNA was not expressed in the brain. PLA2-IIA, PLA2-IV and PLA2-VI mRNAs appeared to be ubiquitously expressed in brain, with relatively similar levels detected in all regions. PLA2-IIC mRNA was expressed in all brain regions but not in any of the peripheral tissues studied. PLA2-V mRNA was found at low levels in most areas of the brain, but at very high levels in the hippocampus. These results indicate that mammalian brain has the potential to express multiple isoforms of PLA2 which could be important given the potential role of these enzyme activities in ischaemic damage and in the regulation of synaptic plasticity.

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase–polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10–12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 μM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Nerve growth factor (NGF) is a mediator of hyperalgesia and has been previously associated with sensory and reflex changes after inflammation of the urinary bladder. A sensitive assay was developed to examine neurotrophin gene expression after bladder inflammation by turpentine, which causes a short-lived inflammatory response. Two hours, but not 6 or 24 h after induction of inflammation, there were significant increases in levels of NGF, brain-derived neurotrophic factor and neurotrophin-3 mRNAs. NGF immunoreactivity was elevated with a similar time course to its mRNA. Our results suggest that during bladder inflammation, endogenous NGF is rapidly up-regulated and released to mediating sensory and reflex changes. Brain-derived neurotrophic factor and neurotrophin-3 may also have a role in the inflammatory response.

Kainate-induced seizure activity causes persistent changes in the hippocampus that include synaptic reorganization and functional changes in the mossy fibers. Using in situ hybridization histochemistry, the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon mRNAs was investigated in the hippocampus of adult rats following seizures induced by a s.c. injection of kainic acid. In CA1 and CA3, we found a significant decrease in PKC gamma mRNA 1 day after kainic acid which persisted for a 2nd day in CA1. None of the other PKC isoform mRNAs were altered in CA1 or CA3. In granule cells, a significant up-regulation specific to PKC epsilon mRNA was observed. One week after kainic acid administration, a marked increase in PKC epsilon immunoreactivity was found that persisted 2 months after kainic acid administration. PKC epsilon immunoreactivity was found associated with mossy fibers projecting to the hilus of the dentate gyrus and to the stratum lucidum of the CA3 field and presumably with the newly sprouted mossy fibers projecting to the supragranular layer. These data provide the first evidence for a long-lasting increase of the PKC epsilon in the axons of granule cells caused by kainate-induced seizures and suggest that PKC epsilon may be involved in the functional and/or structural modifications of granule cells that occur after limbic seizures.

We report the presence in rat spinal cord of a novel neuronal system expressing tyrosine kinase receptor (trkA), the high affinity receptor for nerve growth factor (NGF). TrkA immunoreactive cell bodies were observed in the intermediate grey matter of the spinal cord and were classified into three main groups: central canal cells located dorsolateral to the aqueduct, partition cells located between lamina X, and the lateral border of the intermediate grey, and a morphologically heterogeneous group which included large cells located near the lateral border. In situ hybridization confirmed that cells in all these areas express trkA mRNA. Combined immunofluorescence and retrograde Fluoro-Gold labelling was used to further characterise the projections and neurotransmitter profile of the trkA cells. Although often located in the vicinity of preganglionic cell groups, trkA immunoreactive cells are not themselves preganglionic. Rather, the central canal and partition cells belong to a neurochemically complex cholinergic propriospinal system. Many partition cells coexpress trkA, choline acetyltransferase (ChAT), the low affinity neurotrophin receptor, p75, and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). In contrast, trkA immunoreactive central canal cells express ChAT, but do not express p75 and only a subpopulation express NADPH-d. The large trkA immunoreactive cells located on the lateral border do not express ChAT. TrkA immunoreactive fibres were also present and were located in the dorsal horn, in the dorsal columns, and in a bundle ventral to the aqueduct. However, double labelling revealed that the trkA immunoreactive fibres are not intrinsic but are primary afferent in origin and coexpress p75. The location of this novel trkA neuronal system is consistent with it having a role in the segmental integration of autonomic outflow. NGF could affect this system by modulating neuronal phenotype and/or synaptic efficacy.

Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K(+). In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial:GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.

Astrocyte reactivity is implicated in the neuronal loss underlying Alzheimer's disease. Curcumin has been shown to reduce astrocyte reactivity, though the exact pathways underlying these effects are incompletely understood. Here we investigated the role of the small ubiquitin-like modifier (SUMO) conjugation in mediating this effect of curcumin. In beta-amyloid (Aβ)-treated astrocytes, morphological changes and increased glial fibrillary acidic protein (GFAP) confirmed reactivity, which was accompanied by c-jun N-terminal kinase activation. Moreover, the levels of SUMO-1 conjugated proteins, as well as the conjugating enzyme, Ubc9, were decreased, with concomitant treatment with curcumin preventing these effects. Increasing SUMOylation in astrocytes, by over-expression of constitutively active SUMO-1, but not its inactive mutant, abrogated Aβ-induced increase in GFAP, suggesting astrocytes require SUMO-1 conjugation to remain non-reactive.

Kindling is a well documented model of acquired focal epilepsy and synaptic plasticity in the nervous system. Previous biochemical studies have indicated an increase in mGluR-mediated phosphoinositide hydrolysis in the amygdala or hippocampus of fully kindled animals. In this study we have used in situ hybridisation techniques to examine the mRNA expression of group I metabotropic glutamate receptors (mGluR1 and mGluR5 both linked to phosphoinositide hydrolysis) in the hippocampus of amygdala-kindled animals sacrificed 24 h, 7 days or 28 days following the last electrically evoked stage 5 seizure, and in implanted non-stimulated control rats. Results indicate an initial up-regulation in mGluR1 mRNA (expressed as percentage of control) bilaterally in the DG (35-40%) and CA3 (16-48%), and unilaterally in CA4 (12%) in the 24 h post-kindled group. In kindled animals studied 7 days after the last seizure, these changes were either reduced or had returned to control levels. By 28 days mGluR1 mRNA levels had returned to control levels, with only a persistent increase in expression unilaterally in the DG (14%). In contrast, an initial down-regulation in mGluR5 mRNA was observed bilaterally in CA4 (-45 and -25%) and CA1 (-46 and -45%), and unilaterally in DG and CA3 (-27 and -42% respectively) 24 h after the last kindled seizure. In the 7 and 28 day kindled groups significant alterations in expression of mGluR5 mRNA were still apparent. These data show that the mRNAs for mGluR1 and mGluR5 are differentially regulated by kindling, indicating that the expression of each of these receptors is under independent regulatory control. These perturbations in mRNA expression may contribute to kindling epileptogenesis but are unlikely to account for the maintenance of the kindled state.

Flavonoids exert a multiplicity of neuroprotective actions within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory

Evidence suggests that flavonoid-rich foods are capable of inducing improvements in memory and cognition in animals and humans. However, there is a lack of clarity concerning whether flavonoids are the causal agents in inducing such behavioral responses. Here we show that supplementation with pure anthocyanins or pure flavanols for 6 weeks, at levels similar to that found in blueberry (2% w/w), results in an enhancement of spatial memory in 18 month old rats. Pure flavanols and pure anthocyanins were observed to induce significant improvements in spatial working memory (p = 0.002 and p = 0.006 respectively), to a similar extent to that following blueberry supplementation (p = 0.002). These behavioral changes were paralleled by increases in hippocampal brain-derived neurotrophic factor (R = 0.46, p

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) - Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

domains prevent fibril formation of Ab 40 and A b 42 far below stoichiometric ratio. Kinetic experiments show that BRICHOS prolongs the lag time for fibrillation and the effects on the elongation phase are more prominent for Ab 42 than for A b 40 (Fig. 1). The inhibitory effect is concentration dependent in a quantitative and highly reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase. Circular dichroism spectroscopy and analytical size exclusion chromatography imply that a main fraction of Ab is maintained as unstructured monomer during the extended lag phase in the presence of BRICHOS. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded b-sheet, with inter-hydroxyl distances suited for hydrogen bonding peptides in extended b-conformation. This array is lined with more charged residues in Bri2 than in proSP-C. Conclusions: Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with processes involving aggregated species, most likely fibril-dependent secondary nucleation, which is a major determinant of the length of the lag phase and the sharpness of the kinetic transition for Ab.

Background Riluzole is a neuroprotective drug used in the treatment of motor neurone disease. Recent evidence suggests that riluzole can up-regulate the expression and activity of the astrocyte glutamate transporter, GLT-1. Given that regulation of glutamate transport is predicted to be neuroprotective in Parkinson's disease, we tested the effect of riluzole in parkinsonian rats which had received a unilateral 6-hydroxydopamine injection into the median forebrain bundle. Results Rats were treated with intraperitoneal riluzole (4 mg/kg or 8 mg/kg), 1 hour before the lesion then once daily for seven days. Riluzole produced a modest but significant attenuation of dopamine neurone degeneration, assessed by suppression of amphetamine-induced rotations, preservation of tyrosine hydroxylase positive neuronal cell bodies in the substantia nigra pars compacta and attenuation of striatal tyrosine hydroxylase protein loss. Seven days after 6-hydroxydopamine lesion, reactive astrocytosis was observed in the striatum, as determined by increases in expression of glial fibrillary acidic protein, however the glutamate transporter, GLT-1, which is also expressed in astrocytes was not regulated by the lesion. Conclusions The results confirm that riluzole is a neuroprotective agent in a rodent model of parkinson's disease. Riluzole administration did not regulate GLT-1 levels but significantly reduced GFAP levels, in the lesioned striatum. Riluzole suppression of reactive astrocytosis is an intriguing finding which might contribute to the neuroprotective effects of this drug.

The midbrain periaqueductal grey matter (PAG) has numerous functional roles that include mediating nociceptive inhibition and integrating behavioural and physiological responses to potentially threatening or stressful stimuli. Underlying these behaviours is the diverse interconnectivity of this region, and it is possible that neurochemical subdivisions within the PAG reflect the functional properties of the different PAG regions. In this study, using in situ hybridization, we have investigated the distribution in the rat PAG of the messenger ribonucleic acids (mRNAs) encoding seven neuropeptides: enkephalin (ENK), substance P (SP), somatostatin (SST), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Each peptide mRNA had a distinct topographical distribution in the PAG. Preproenkephalin A (ENK) mRNA-expressing cells were found at all levels of the PAG in three distinct longitudinal columns. Preprotachykinin A (SP)-expressing cells were found at all levels of the PAG, principally in the Edinger-Westphal nucleus and the lateral and dorsal PAG. There was a column of neurons producing mRNA-encoding somatostatin that extended along the rostrocaudal extent of the ventrolateral PAG; there were also labelled cells in the dorsal and dorsolateral subdivisions at some levels of the PAG. Galanin mRNA-producing neurones were limited to the dorsal raphe nucleus and to a second population in the ventral border of the aqueduct. VIP mRNA-producing neurones were found in very localized regions of the PAG, including the cell-sparse region immediately ventral to the aqueduct and the ventral part of the dorsal raphe nucleus. NPY mRNA-producing neurones were localized mainly in some cells of the Edinger-Westphal nucleus and dorsal raphe nucleus. CGRP mRNA-expressing neurons were limited to the oculomotor and trochlear nucleus. The results showed a topographical distribution of neuropeptides over the rostrocaudal extent of the PAG that is compatible with the emerging theory that the anatomical and functional specificity of the PAG is expressed in the form of longitudinally arranged neuronal columns that extend for varying distances along the rostrocaudal axis of the midbrain PAG.

The effect of repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) on the expression of tyrosine hydroxylase and preprocholecystokinin (CCK) messenger RNAs in substantia nigra was examined by in situ hybridisation histochemistry. Sections hybridised with 35S-labelled oligonucleotides were subjected to computerised image analysis to determine the density of silver grains above positively labelled cells as an index of steady state mRNA levels. In the substantia nigra pars compacta, CCK mRNA levels were significantly reduced in drug-treated animals 24 h and at 2 weeks after the last dose of MDMA (10 mg/kg i.p., twice daily for 4 days). In the same animals, MDMA caused no change in the level of tyrosine hydroxylase mRNA in this brain region. The results show that MDMA can produce changes in dopamine neurones. Furthermore, since tyrosine hydroxylase and cholecystokinin are co-expressed in substantia nigra pars compacta, these results suggest that the expression of the tyrosine hydroxylase and CCK genes are regulated independently.

In situ hybridization was used to measure the effects of chronic fenfluramine administration on serotonin transporter messenger RNA expression in cells of the dorsal raphe nucleus complex. Fenfluramine produced a significant, but transient, down-regulation of serotonin transporter mRNA in cells which lie in the ventral portion of the dorsal raphe nucleus, but not in the dorsal part of the dorsal raphe nucleus. Our findings suggest that cells which lie in the ventral part of the dorsal raphe nucleus are more sensitive to the effects of chronic fenfluramine administration, but that fenfluramine does not cause long-term changes in gene expression in serotonin cell bodies.

Preprocholecystokinin (CCK) mRNA expression was measured in a subset of hippocampal interneurones after transection of afferent septohippocampal fibres in the fimbria fornix. Two weeks after the lesions were made, CCK mRNA levels were significantly lower in these neurones on the side ipsilateral to the lesion compared with equivalent neurones on the unlesioned side. These findings suggest that axotomizing lesions can change gene expression in denervated target cells, and that CCK mRNA levels in hippocampal interneurones may be modulated by afferent septohippocampal input.

Flavonoid-rich foods have been shown to be able to reverse age-related cognitive deficits in memory and learning in both animals and humans. However, to date, there have been only a limited number of studies investigating the effects of flavonoid-rich foods on cognition in young/healthy animals. The aim of this study was to investigate the effects of a blueberry-rich diet in young animals using a spatial working memory paradigm, the delayed non-match task, using an eight-arm radial maze. Furthermore, the mechanisms underlying such behavioural effects were investigated. We show that a 7-week supplementation with a blueberry diet (2 % w/w) improves the spatial memory performance of young rats (2 months old). Blueberry-fed animals also exhibited a faster rate of learning compared to those on the control diet. These behavioural outputs were accompanied by the activation of extracellular signal-related kinase (ERK1/2), increases in total cAMP-response element-binding protein (CREB) and elevated levels of pro- and mature brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in hippocampal CREB correlated well with memory performance. Further regional analysis of BDNF gene expression in the hippocampus revealed a specific increase in BDNF mRNA in the dentate gyrus and CA1 areas of hippocampi of blueberry-fed animals. The present study suggests that consumption of flavonoid-rich blueberries has a positive impact on spatial learning performance in young healthy animals, and these improvements are linked to the activation of ERK–CREB–BDNF pathway in the hippocampus.

We describe a novel procedure for combining immunocytochemistry with in situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridization and is particularly suitable for antigens which are labile or sensitive to in situ hybridization processing. We have evaluated the technique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing 35S-labelled oligonucleotide probes. Successful double labelling was obtained and showed that galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive cells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic cell population. Double-labelled preparations produced using this technique can be conveniently viewed using epipolarised combined with epifluorescent illumination. Careful analysis of procedural variables revealed that it is not possible to carry out high-sensitivity 5-HT immunocytochemistry following in situ hybridization. The immunostaining is much poorer on slide-mounted sections than on free-floating sections, and 5-HT appears to be lost during the in situ hybridization steps of dehydration/delipidation and incubation in hybridization buffer. The procedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.

Using in situ hybridisation with an oligonucleotide probe, the regional distribution of GABA transporter-1 messenger RNA was determined in the adult rat CNS. Overall, the distribution of GABA transporter-1 mRNA was similar to that of glutamate decarboxylase-67 mRNA, consistent with an expression in GABA neurones. However, in cerebellar cortex, Purkinje cells which express high levels of glutamate decarboxylase-67 mRNA did not express GABA transporter-1 mRNA, whereas Bergmann glia expressed GABA transporter-1 mRNA at high levels. In some other brain regions, including the inferior colliculus and reticular thalamus, there was no GABA transporter-1, although there were neurones which expressed glutamate decarboxylase-67 messenger RNA at high levels.

Using in situ hybridisation, the effects of acute and chronic diazepam administration and diazepam withdrawal on preprocholecystokinin (CCK) mRNA levels in discrete regions of rat brain were determined. In cerebral cortex and a subpopulation of hippocampal neurones, CCK mRNA levels were increased after a single injection of diazepam and 24 h after withdrawal from chronic diazepam treatment, but not after chronic diazepam treatment. These results show that, in some neuronal groups, CCK mRNA expression is regulated by benzodiazepines, although there is no clear link between CCK mRNA levels and anxiety state.