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Genomic variation can impact normal biological function in complex ways and so understanding variant effects requires a broad range of data to be coherently assimilated. Whilst the volume of human variant data and relevant annotations has increased, the corresponding increase in the breadth of participating fields, standards and versioning mean that moving between genomic, coding, protein and structure positions is increasingly complex. In turn this makes investigating variants in diverse formats and assimilating annotations from different resources challenging. ProtVar addresses these issues to facilitate the contextualization and interpretation of human missense variation with unparalleled flexibility and ease of accessibility for use by the broadest range of researchers. By precalculating all possible variants in the human proteome it offers near instantaneous mapping between all relevant data types. It also combines data and analyses from a plethora of resources to bring together genomic, protein sequence and function annotations as well as structural insights and predictions to better understand the likely effect of missense variation in humans. It is offered as an intuitive web server https://www.ebi.ac.uk/protvar where data can be explored and downloaded, and can be accessed programmatically via an API., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We perform saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identify 3432 functionally abnormal variants, in three distinct classes. We train a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants can account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation., (© 2023. The Author(s).)

Constrained Coding Regions (CCRs) in the human genome have been derived from DNA sequencing data of large cohorts of healthy control populations, available in the Genome Aggregation Database (gnomAD) [1]. They identify regions depleted of protein-changing variants and thus identify segments of the genome that have been constrained during human evolution. By mapping these DNA-defined regions from genomic coordinates onto the corresponding protein positions and combining this information with protein annotations, we have explored the distribution of CCRs and compared their co-occurrence with different protein functional features, previously annotated at the amino acid level in public databases. As expected, our results reveal that functional amino acids involved in interactions with DNA/RNA, protein-protein contacts and catalytic sites are the protein features most likely to be highly constrained for variation in the control population. More surprisingly, we also found that linear motifs, linear interacting peptides (LIPs), disorder-order transitions upon binding with other protein partners and liquid-liquid phase separating (LLPS) regions are also strongly associated with high constraint for variability. We also compared intra-species constraints in the human CCRs with inter-species conservation and functional residues to explore how such CCRs may contribute to the analysis of protein variants. As has been previously observed, CCRs are only weakly correlated with conservation, suggesting that intraspecies constraints complement interspecies conservation and can provide more information to interpret variant effects., Competing Interests: Declaration of Competing Interest, (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.

VarSite is a web server mapping known disease-associated variants from UniProt and ClinVar, together with natural variants from gnomAD, onto protein 3D structures in the Protein Data Bank. The analyses are primarily image-based and provide both an overview for each human protein, as well as a report for any specific variant of interest. The information can be useful in assessing whether a given variant might be pathogenic or benign. The structural annotations for each position in the protein include protein secondary structure, interactions with ligand, metal, DNA/RNA, or other protein, and various measures of a given variant's possible impact on the protein's function. The 3D locations of the disease-associated variants can be viewed interactively via the 3dmol.js JavaScript viewer, as well as in RasMol and PyMOL. Users can search for specific variants, or sets of variants, by providing the DNA coordinates of the base change(s) of interest. Additionally, various agglomerative analyses are given, such as the mapping of disease and natural variants onto specific Pfam or CATH domains. The server is freely accessible to all at: https://www.ebi.ac.uk/thornton-srv/databases/VarSite., (© 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Motivation: Understanding the protein structural context and patterning on proteins of genomic variants can help to separate benign from pathogenic variants and reveal molecular consequences. However, mapping genomic coordinates to protein structures is non-trivial, complicated by alternative splicing and transcript evidence., Results: Here we present VarMap, a web tool for mapping a list of chromosome coordinates to canonical UniProt sequences and associated protein 3D structures, including validation checks, and annotating them with structural information., Availability and Implementation: https://www.ebi.ac.uk/thornton-srv/databases/VarMap., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)

We estimated the genome-wide contribution of recessive coding variation in 6040 families from the Deciphering Developmental Disorders study. The proportion of cases attributable to recessive coding variants was 3.6% in patients of European ancestry, compared with 50% explained by de novo coding mutations. It was higher (31%) in patients with Pakistani ancestry, owing to elevated autozygosity. Half of this recessive burden is attributable to known genes. We identified two genes not previously associated with recessive developmental disorders, KDM5B and EIF3F , and functionally validated them with mouse and cellular models. Our results suggest that recessive coding variants account for a small fraction of currently undiagnosed nonconsanguineous individuals, and that the role of noncoding variants, incomplete penetrance, and polygenic mechanisms need further exploration., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes. These processes include both the initial emergence of functional structures in the presence of the mineral and the subsequent transition away from the mineral-associated niche. The initial evolution of function depends upon the number and distribution of sequences capable of functioning in the presence of the mineral, and the transition to new environments depends upon the overlap between sequences that evolve on the mineral surface and sequences that can perform the same functions in the mineral's absence. To examine these processes, we evolved self-cleaving ribozymes in vitro in the presence or absence of Na-saturated montmorillonite clay mineral particles. Starting from a shared population of random sequences, RNA populations were evolved in parallel, along separate evolutionary trajectories. Comparative sequence analysis and activity assays show that the impact of this clay mineral on functional structure selection was minimal; it neither prevented common structures from emerging, nor did it promote the emergence of new structures. This suggests that montmorillonite does not improve RNA's ability to evolve functional structures; however, it also suggests that RNAs that do evolve in contact with montmorillonite retain the same structures in mineral-free environments, potentially facilitating an evolutionary transition away from a mineral-associated niche., (© 2016 Stephenson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The importance of elucidating the three dimensional structures of RNA molecules is becoming increasingly clear. However, traditional protein structural techniques such as NMR and X-ray crystallography have several important drawbacks when probing long RNA molecules. Single molecule Förster resonance energy transfer (smFRET) has emerged as a useful alternative as it allows native sequences to be probed in physiological conditions and allows multiple conformations to be probed simultaneously. This review serves to describe the method of generating a three dimensional RNA structure from smFRET data from the biochemical probing of the secondary structure to the computational refinement of the final model., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Similarities and differences between amino acids define the rates at which they substitute for one another within protein sequences and the patterns by which these sequences form protein structures. However, there exist many ways to measure similarity, whether one considers the molecular attributes of individual amino acids, the roles that they play within proteins, or some nuanced contribution of each. One popular approach to representing these relationships is to divide the 20 amino acids of the standard genetic code into groups, thereby forming a simplified amino acid alphabet. Here, we develop a method to compare or combine different simplified alphabets, and apply it to 34 simplified alphabets from the scientific literature. We use this method to show that while different suggestions vary and agree in non-intuitive ways, they combine to reveal a consensus view of amino acid similarity that is clearly rooted in physico-chemistry.

We have detected a concentration of boron in martian clay far in excess of that in any previously reported extra-terrestrial object. This enrichment indicates that the chemistry necessary for the formation of ribose, a key component of RNA, could have existed on Mars since the formation of early clay deposits, contemporary to the emergence of life on Earth. Given the greater similarity of Earth and Mars early in their geological history, and the extensive disruption of Earth's earliest mineralogy by plate tectonics, we suggest that the conditions for prebiotic ribose synthesis may be better understood by further Mars exploration.

HIV-1 genomic RNA has a noncoding 5' region containing sequential conserved structural motifs that control many parts of the life cycle. Very limited data exist on their three-dimensional (3D) conformation and, hence, how they work structurally. To assemble a working model, we experimentally reassessed secondary structure elements of a 240-nt region and used single-molecule distances, derived from fluorescence resonance energy transfer, between defined locations in these elements as restraints to drive folding of the secondary structure into a 3D model with an estimated resolution below 10 Å. The folded 3D model satisfying the data is consensual with short nuclear-magnetic-resonance-solved regions and reveals previously unpredicted motifs, offering insight into earlier functional assays. It is a 3D representation of this entire region, with implications for RNA dimerization and protein binding during regulatory steps. The structural information of this highly conserved region of the virus has the potential to reveal promising therapeutic targets., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although numerous hypotheses have been proposed to explain geophagy, the primary driver of this behaviour remains elusive. Supplementation of scarce nutrients is one commonly cited explanation. We examined the element concentration of three licks relative to adjacent topsoils to infer the possible reasons for geophagy at Loskop Dam Nature Reserve. Lick samples had greater concentrations of B, Co, Zn, Se, Mo and Mn (Loskop Main Lick); Cu (Klopperskloof Lick); and Na (Klopperskloof Lick and Rhenosterhoek Lick) than those of adjacent topsoil. We suggest that supplementation with all or some of these nutrients is a likely driver of geophagy in this fenced reserve, with different licks providing herbivores with different suites of nutrients.

Increased production and deposition of amyloid beta-protein (Abeta) are believed to be key pathogenic events in Alzheimer's disease. As such, routes for lowering cerebral Abeta levels represent potential therapeutic targets for Alzheimer's disease. X11beta is a neuronal adaptor protein that binds to the intracellular domain of the amyloid precursor protein (APP). Overexpression of X11beta inhibits Abeta production in a number of experimental systems. However, whether these changes to APP processing and Abeta production induced by X11beta overexpression also induce beneficial effects to memory and synaptic plasticity are not known. We report here that X11beta-mediated reduction in cerebral Abeta is associated with normalization of both cognition and in vivo long-term potentiation in aged APPswe Tg2576 transgenic mice that model the amyloid pathology of Alzheimer's disease. Overexpression of X11beta itself has no detectable adverse effects upon mouse behaviour. These findings support the notion that modulation of X11beta function represents a therapeutic target for Abeta-mediated neuronal dysfunction in Alzheimer's disease.

The medial septal area of conscious rats was stimulated through previously implanted electrodes at a frequency of 7.7 Hz for 20 min each day for 7 days to evoke rhythmic slow activity in CA1 at a similar frequency to spontaneous theta. Two weeks later in the anaesthetized rats the effects of a single subcutaneous injection of nicotine (0.4 mg/kg) on fEPSPs, evoked in the dentate gyrus by separate stimulation of the MPP and LPP, were studied and compared with those obtained in controls. Nicotine increased the firing of locus coeruleus neurons and the slope of the fEPSPs evoked by LPP stimulation, but not by MPP stimulation. Prior theta driving considerably increased the effect of nicotine on the responses evoked by stimulation of the MPP and abolished the nicotine-induced potentiation of the responses evoked by stimulation of the LPP. The results are attributed to theta driving increasing the amount of noradrenaline released by nicotine and to noradrenaline producing a beta-adrenoceptor long-lasting potentiation at the medial perforant path synapse and a long-lasting depression at the lateral perforant path synapse.

This study investigated the response of hippocampal RSA, recorded from electrodes in CA1 and the contralateral dentate gyrus of urethane-anaesthetized rats, to atropine sulphate administered at 15 min intervals in a cumulative dose-response schedule (1, 3, 10, 50 and 50 mg x kg(-1) i.p.). The power of CA1 and dentate gyrus RSA in the 3-7 Hz band was increased after administering the first 3 doses of atropine (1, 3 and 10 mg x kg(-1) cumulatively) in rats held in the stereotaxic frame or removed from the frame and given electrical sensory stimulation to the base of the tail. This increase in RSA was dependent on sensory input, since it was not seen in animals outside the frame unless sensory stimulation was given, and it was abolished by increasing the dose of atropine (an additional 50 and 50 mg x kg(-1) cumulatively). Methylatropine (6 mg x kg(-1) i.p.) did not increase RSA power. The biphasic effect of atropine on sensory-evoked hippocampal RSA activity may be explained by differential effects at pre- and post-synaptic sites e.g. in the septo-hippocampal system or on pathways processing sensory information.

The medial septal area of conscious rats was stimulated through previously implanted electrodes at a frequency of 7.7 Hz for 20 min each day for 7 days to evoke rhythmic slow activity in CA1 at a similar frequency to spontaneous theta. Two weeks later in the anaesthetized rats the effects of a single subcutaneous injection of nicotine (0.4 mg x kg(-1)) on fEPSPs, evoked in the dentate gyrus by separate stimulation of the MPP and LPP, were studied and compared with those obtained in controls. Nicotine increased the firing of locus coeruleus neurones and the slope of the fEPSPs evoked by LPP stimulation, but not by MPP stimulation. Prior theta driving considerably increased the effect of nicotine on the responses evoked by stimulation of the MPP and abolished the nicotine-induced potentiation of the responses evoked by stimulation of the LPP. The results are attributed to theta driving increasing the amount of noradrenaline released by nicotine and to noradrenaline producing a beta-adrenoceptor long-lasting potentiation at the medial perforant path synapse and a long-lasting depression at the lateral perforant path synapse.

The combination of thoracic aortic pseudoaneurysm and left ventricular aneurysm resulting from a single traumatic incident is an exceedingly rare occurrence. We present a case of a 10-year-old girl who sustained significant blunt trauma to the chest after being involved in a rollover motor vehicle accident. The child underwent immediate repair of a transected aortic arch. An inferior wall left ventricular aneurysm developed 3 weeks later, and the patient underwent successful repair of the left ventricular aneurysm and a damaged mitral valve. The use of fast multidetector row CT, cardiac MRI, and echocardiography have improved our ability to diagnose these types of injuries and accurately image their anatomic relationships in the acute and perioperative settings.

Rationale: Clozapine-like atypical antipsychotic drugs, such as olanzapine, risperidone and sertindole, bind most strongly to 5-HT(2A) receptors, which may contribute to their antipsychotic effects. Antipsychotic drugs, such as clozapine and haloperidol, have been found to enhance latent inhibition (LI) in humans and rats. LI is a process of learning to ignore irrelevant stimuli that is disrupted in acute, positive-symptom schizophrenia, and can be modelled in animals., Objectives: The aim of this study was to determine the effects of two selective 5-HT(2A) receptor antagonists, SR 46,349B and ICI 169,369, on LI, as a test of their antipsychotic potential., Methods: Doses of the 5-HT(2A) receptor antagonists that were sufficient for receptor blockade were determined in 5-HT behavioural syndrome tests. SR 46,349B and ICI 169,369 were then tested for enhancement of LI and reversal of amphetamine-induced attenuation of LI in a conditioned suppression paradigm., Results: SR 46,349B (0.6-2.4 mg kg(-1) i.p.) and ICI 169,369 (10-40 mg kg(-1) i.p.) antagonised 5-hydroxytryptophan (5-HTP)-induced head twitches and wet dog shakes, which are mediated by 5-HT(2A) receptors, but had no effect on mCPP-induced hypolocomotion, which is mediated by 5-HT(2C) receptors. Neither SR 46,349B (1.2 mg kg(-1) i.p.) nor ICI 169,369 (40 mg kg(-1) i.p) affected 8-hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT)-induced forepaw treading, suggesting that they were not in vivo 5-HT(1A) receptor antagonists. SR 46,349B (2.4 mg kg(-1) i.p.) and ICI 169,369 (40 mg kg(-1) i.p.) enhanced LI when given at both the pre-exposure and conditioning stages of the paradigm, but not when given at either pre-exposure or conditioning only. Both drugs also reversed the disruption of LI induced by D-amphetamine (1 mg kg(-1) i.p.)., Conclusions: The profile of SR 46,349B and ICI 169,369 in LI differs from that of clozapine and haloperidol in LI, which both enhance LI when given only at the conditioning stage of the paradigm.

Latent inhibition (LI) is a model of attention, which is a cognitive process that can be modulated by stressors such as chronic intermittent broadband noise, e.g. caused by building work, which is particularly stressful to rats. The aim of this study was to analyse the effect of chronic noise stress, caused by a building project, on LI, and its interaction with SR 46,349B, a 5-HT2A receptor antagonist. Control groups from LI experiments conducted during periods of chronic intermittent noise were compared with control groups from LI experiments conducted in normal quiet conditions. The interaction of SR 46,349B with the effects of chronic noise stress was then tested. Chronic intermittent noise attenuated LI, an effect which was partially reversed by SR 46,349B, 2.4 mg/kg i.p. Attenuation of LI by chronic intermittent noise and reversal of this effect by SR 46,349B support suggestions that stress can modulate attention and that 5-HT2A receptors are involved in mediating the effects of chronic stress.

The functional connectivity of an embryonic graft implanted into the lesioned somatosensory cortex and the effect of DSP4 (a selective noradrenergic neurotoxin to noradrenergic terminals) and dizolcipine (a non-competitive NMDA receptor antagonist), was studied electrophysiologically. The forepaw representational area of the rat primary somatosensory cortex was lesioned unilaterally and, 3-4 weeks later, tissue from the same region of E19 rat embryos was implanted into the cavity. At 7-9 months later, the rats were anaesthetized and single unit activity was recorded from the grafts in response to contralateral forepaw, ipsilateral hindpaw and contralateral hindpaw stimulation and compared with that obtained in control rats, in rats pretreated with dizolcipine immediately after lesioning and in rats given DSP4 24 h before transplantation. Neurones within the graft were integrated into the host brain and developed a pattern of representation similar to that of intact rats, but with a reduced proportion of neurones exhibiting short-latency response to contralateral forepaw stimulation and an increased proportion responding to stimulation of more than one paw. Pretreatment with dizolcipine did not increase short-latency responses to stimulation of contralateral forepaw stimulation however pretreatment with DSP4 reduced such responses and increased proportion of inhibitory responses. It was concluded that the noradrenergic system plays an important role in establishing host-graft connectivity. The importance of further pharmacological studies on host-graft connectivity and the relation of such connections to neural plasticity were discussed.

Following transection of peripheral nerve, rats exhibit autotomy, which is considered to be the animal model of postdenervation pain syndrome. It has been suggested that phantom limb pain is a result of peripheral denervation leading to reorganization of somatosensory pathways, particularly in the cerebral cortex, which is shown to depend upon central noradrenergic activity. In this study, sciatic and saphenous nerves were sectioned in the left hindpaw of 30 adult rats resulting in complete loss of pain sensation in the hindpaw. A group of rats received normal saline, compared to another group which received N-(2-) Chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) injection 24 h prior to transection. The latter group was also compared to a third group whose central noradrenergic system were also blocked by bilateral injection of 6-OHDA into the ascending noradrenergic bundle 1 week prior to transection. A fourth group received contralateral cortical ablation in addition to peripheral nerve transection and was compared to the first group whose cortex remained intact. The animals were observed daily for 60 days and autotomy was scored in accordance to the system of Wall et al. After 1 week, control animals began to exhibit autotomy. In contrast, autotomy was absent in rats treated with DSP4, similar to rats which received 6-OHDA. Rats which had contralateral cortical ablation showed a considerably delayed onset of autotomy and a reduction in final autotomy scores. We conclude that autotomy, as a model of postdenervation pain syndrome, can be prevented by blockade of noradrenergically mediated cortical reorganization. The clinical implications of this finding are discussed.

The reorganisation of primary somatosensory cortex that occurs after lesioning the corresponding cortex of the contralateral hemisphere in rat has been termed, 'transhemispheric cortical reorganisation'. Cholinergic and noradrenergic innervations are hypothesized to be involved in cortical plasticity. The present study investigated the change in responses of somatosensory neurones in the hindpaw representation area to muscarinic cholinoceptor and beta-adrenoceptor receptor stimulation, by iontophoretic application of acetylcholine, noradrenaline, propranolol and atropine, during the process of transhemispheric cortical reorganization at 3-4 days and at 20-21 days after lesioning the corresponding area in the contralateral hemisphere. Most neurones in control rats showed excitatory atropine-sensitive responses to acetylcholine, and inhibitory propranolol-sensitive responses to noradrenaline. A marked reduction in neurones exhibiting muscarinic responses (from 69% to 22%) and beta-noradrenoceptor-mediated responses (from 62% to 24%) were seen in rats 3-4 days post lesion. The proportion of neurones responding had recovered by 3 weeks but the direction of the responses had changed with muscarinic response becoming predominantly inhibitory and beta-noradrenoceptor responses predominantly excitatory. It is concluded that transhemispheric cortical reorganization involves both receptor types and that the reciprocal changes at different stages after injury maintain cortical plasticity.

Responses of single units in the hindpaw representational area of the left primary somatosensory cortex to electrical stimulation of both hindpaws and the right forepaw were recorded under urethane anaesthesia in three groups of adult male rats: a control group and two groups in which the right hindpaw representational area had been ablated 3-4 weeks previously, immediately after intraperitoneal injection of saline vehicle or DSP4, to destroy cortical noradrenergic terminals arising from the locus coeruleus. The lesion increased the overall number of neurones responding within 500 ms after the stimulation of the contralateral hindpaw (from 64 to 91%), and the proportion exhibiting short-latency response increased from 41 to 61%. Interestingly, the proportion of neurones with bilateral representation increased from 3 to 10% after the cortical lesioning. The changes were prevented by injection of DSP4 prior to lesioning and therefore depended on an intact central noradrenergic system. The increase in bilateral representation could not have been due to direct interhemispheric connections between corresponding representational areas because it occurred after lesioning of the homologous area in the contralateral hemisphere. The phenomenon was termed 'transhemispheric reorganization' and because it was somatotopically oriented (e.g. to either hindpaw); its function may be to ensure that when a sensory cortical area is damaged, its basic sensory functions are 'taken over' by the corresponding contralateral area.

The effects of S-alpha-amino-3-hydroxy-4-isoxozolepropionic acid (AMPA) lesions of the nucleus basalis magnocellularis on the M1/M2 nature of the responses of somatosensory cortical neurones to acetylcholine (ACh) in Sprague-Dawley rats were investigated by iontophoretic application and extracellular single unit recording. The responses were characterised using pirenzepine, an M1 receptor antagonist, and gallamine, an M2 antagonist. Eighty two neurones in control and 94 neurones in lesioned animals were studied. In control animals, 37% of responses to ACh were sensitive to pirenzepine, gallamine or to both antagonists. This increased to 62% in lesioned animals, the proportions of pirenzepine- and gallamine-sensitive responses remaining unchanged. These results provide the first electrophysiological confirmation that both pirenzepine- and gallamine-sensitive (M1 and M2) receptors occur postsynaptic to afferent cholinergic terminals and that their postsynaptic stimulation may produce both inhibition and excitation.

A challenge dose of nicotine (0.4 mg kg-1 s.c.) produced a long-lasting potentiation (LLP) of field EPSPs evoked in the dentate gyrus by stimulation of the medial perforant path of urethane-anaesthetized rats primed four weeks previously with seven daily injections of nicotine (0.8 mg kg-1 s.c.). The same dose of nicotine did not alter EPSPs evoked in rats primed with saline vehicle. Systemic injection of either mecamylamine or propranolol, 30 min before nicotine challenge, prevented induction of LLP without affecting baseline EPSPs but neither drug affected LLP when given after it was established. Since the nicotine-priming regime is known to increase tyrosine hydroxylase activity and hippocampal noradrenaline release in response to nicotine challenge, the results suggest that systemic injection of nicotine released sufficient noradrenaline in nicotine-primed rats to induce a beta-adrenoceptor-mediated LLP.

Responses were evoked in the hippocampal CA1 field by stimulating the medial septal area (MSA) and ventral hippocampal commissure (VHC) of urethane-anaesthetized rats. Tetanic stimulation of the MSA resulted in a homosynaptic long-term potentiation (LTP) of a slow synaptic response in the hippocampus, and heterosynaptic LTP of the evoked VHC response. Both forms of LTP were blocked by MK801. Tetanization of the VHC resulted in homosynaptic LTP but did not produce heterosynaptic LTP of the evoked MSA response. Atropine pretreatment increased commissural homosynaptic LTP and unveiled a heterosynaptic LTP of MSA-evoked responses. In contrast, atropine pretreatment partially blocked the homosynaptic LTP produced by tetanization of the MSA and prevented the heterosynaptic LTP of VHC-evoked responses.

The phenomenon of long-term potentiation is widely used as an experimental model of memory. An approach that has been used to study its underlying mechanisms is to analyse its interaction with presynaptic paired-pulse facilitation. Several studies found no evidence for an interaction in the CA1 hippocampal area, whereas other data, for example from quantal analysis, suggested that presynaptic mechanisms contribute to the maintenance of long-term potentiation. In the present study, initial slopes of field potentials in area CA1 were measured in rat hippocampal slices. "Conventional" long-term potentiation was induced by high-frequency (100 Hz) afferent tetanization of the testing input. "Associative" long-term potentiation was induced by combining lower frequency (40 Hz) tetanization of a testing input with high-frequency tetanization of a second input. The paired-pulse facilitation ratio decreased in the majority of experiments in which long-term potentiation was induced conventionally, but it decreased, increased or did not change after inducing associative potentiation. Decreases in the paired-pulse facilitation correlated inversely with the initial (pre-tetanic) facilitation ratio. A more detailed regression analysis suggests that this correlation results from two other correlations: (i) that between changes in paired-pulse facilitation and the magnitude of long-term potentiation, and (ii) that between initial paired-pulse facilitation and the magnitude of long-term potentiation. The first correlation prevailed during the initial 10 min following tetanization, while the second prevailed 40-60 min later. A post-tetanic decrease in paired-pulse facilitation is evidence for an involvement of presynaptic mechanisms in the maintenance of long-term potentiation. The lack of significant changes in some studies could be due to the inclusion in the analyses of experiments with long-term potentiation of small magnitude, in which changes in paired-pulse facilitation ratios would have been inconsistent. The present study suggests that the early (10-20 min) and late (40-50 min) phases of long-term potentiation were mediated by different mechanisms, with a mixture of these mechanisms during the intermediate period. On the basis of the present and previous studies, the following scheme of involvement of several mechanisms in long-term potentiation maintenance is proposed. The early phase includes two major mechanisms: an increase in the probability of transmitter release, leading to an apparent increase in the number of effective release sites, and an increase in efficacy of one transmitter quantum, probably due to an increased number of postsynaptic receptors. The later phase of long-term potentiation is attributed to an increase in the number of transmitter zones, presumably due to structural modifications.

This study compared the effects of dopamine receptor stimulation in the entorhinal cortex on dopamine release in the nucleus accumbens, measured by in vivo microdialysis in conscious Sprague-Dawley rats, with and without oestradiol and progesterone priming. Nonselective dopamine receptor stimulation with apomorphine reduced dopamine release in the nucleus accumbens, an effect which was prevented by injection of cis-flupenthixol into the entorhinal cortex. Selective D1 receptor stimulation with SKF38393 increased dopamine release, whereas selective D2 receptor stimulation with quinpirole did not affect dopamine release. Combined administration of oestradiol and progesterone potentiated the response to apomorphine and prevented the response to SKF38393. The effects of single hormone administration on the response to apomorphine suggested that the modulation was primarily due to oestradiol enhancing effects of progesterone. Experiments with high [K+] suggested these hormonal effects were exerted predominantly in the entorhinal cortex. The present experiments have demonstrated that dopaminergic modulation of transmission in a cortico-striatal loop linking temporal and prefrontal cortex is regulated by oestradiol and progesterone. Dysfunction in this system in humans may give rise to affective and cognitive symptoms which may, if initiated by a postpartum fall in oestrogen and progesterone concentrations, constitute the core pathophysiology of puerperal psychosis.

Unilateral AMPA lesions of the nucleus basalis magnocellularis (nbm) produced a nearly complete loss of cholinergic markers in the ipsilateral frontal and parietal cortices with no recovery at 6 months. The loss was associated with compensatory increases in AChE-positive fibre density in the contralateral cortex, in ipsilateral cortical regions not receiving their cholinergic innervation from the nbm and in the size of cholinergic magnocellular neurones in the contralateral nbm. The hypertrophy and increase in AChE-positive fibre density were apparent at 4-6 weeks after lesion and increased with time. Cholinergic transplants to cholinergically deafferented cortex prevented development of the compensatory increases in AChE-positive fibre density and restored AChE-positive fibre density and ChAT activity to control levels in ipsilateral cholinergically deafferented regions, partially after 6-8 weeks and completely after 6 months. In contrast, when cholinergic grafts were placed into unlesioned cortex, axonal outgrowth was localized to the vicinity of the transplant and did not develop with time. These results support the concept that vacant synapses promote and direct axonal outgrowth from transplanted neurones and that grafted cholinergic neurones integrate into the lesioned forebrain cholinergic projections system and prevent the lesion-induced changes in AChE-positive fibre density and ChAT activity.

Unilateral S-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) lesions of the nucleus basalis magnocellularis (nbm), which produced persistent and extensive ChAT-positive cell loss within the nbm and depletion of cortical cholinergic markers in the frontal cortex, increased both the number and sensitivity of individual frontal cortical neurones responding to iontophoretic administration of ACh. The lesion also increased the sensitivity of individual neurones to carbachol but the increase in the number of neurones responding to carbachol was transient and had returned to normal 4 weeks after lesion. The sensitivity of individual neurones to glutamate was unchanged by the lesion. The percentage of cortical neurones responding to ACh, but not the sensitivity of individual neurones was restored to the prelesion level, 6-8 weeks after cholinergic transplants to the lesioned frontal cortex; cholinergic transplants to the more distant parietal cortex were only effective after 6 months whereas noncholinergic transplants were ineffective at both time intervals. Cholinergic transplants placed in the frontal cortex 6-8 weeks or 6 months before nbm lesion offered some protection from the effects of the lesion, particularly at 6 months but were ineffective when placed into the parietal cortex. Lesion of the nbm also reduced basal firing rate of spontaneously active neurones and this was not restored by any of the transplants. The results are discussed in the light of quantitative measurements of acetylcholinesterase-positive fibre outgrowth from the transplant into the recording area, which are described in the preceding manuscript [20].

The previous articles in this series [4,9] have shown that unilateral AMPA lesions of the nucleus basalis magnocellularis (nbm) produced widespread morphological and functional changes to the forebrain cholinergic projection system that could be reversed by transplants of fetal cholinergic tissue. At earlier postgraft time points, the effects of cholinergic grafts were specific to the neocortical region (frontal or parietal cortex) into which the grafts were targeted. Here we report that nbm lesion-induced spatial learning and memory deficits in the Morris water maze were reversed at 6-8 weeks postsurgery only by cholinergic grafts placed in the frontal cortex or frontal and parietal cortices combined. Similar grafts to parietal cortex only and noncholinergic fetal transplants to any cortical site were ineffective. In contrast, using separate groups of animals, deficits in sensorimotor function could be reversed in only one measure (open field turning) by cholinergic transplants targeted to the parietal (somatosensory) cortex or frontal and parietal cortex combined. These behavioural dissociations demonstrate that the frontal cortical cholinergic innervation from the nbm is necessary for effective spatial cognitive performance.

The activity of single neurones evoked by electrical stimulation of the paws was recorded from the central forepaw and hindpaw receptive fields of the primary somatosensory cortex (Sm1) in anaesthetized rats. In the forepaw receptive area (FRA), 44% of neurones showed pyramidal responses to stimulation of their central receptive fields (CRF) compared with 13% of neurones in the hindpaw receptive area (HRA). The neurones were located primarily in layers IV and V. A number of neurones in superficial layers of the hindpaw region, but not in the forepaw region, had either bilateral or exclusively ipsilateral receptive fields. It was concluded that neurones with bilateral hindpaw receptive fields form a separate population of neurones which are organized somatopically.

The effects of chronic nicotine treatment and of unilateral AMPA lesion of the nucleus basalis magnocellularis (nbm) on the sensitivity of frontal cortical neurones to iontophoretically applied nicotine were studied. Chronic nicotine treatment increased the number of [3H]nicotine binding sites from 2.9 to 3.9 pmol g-1 wet weight, and increased the proportion of cortical neurones responding to nicotine from 32.3% to 60.0%. After unilateral nbm lesions, the densities of AChE-positive fibers and [3H]nicotine binding sites were reduced by approximately 97% and 55%, respectively, and the proportion of neurones responding to nicotine increased from 32.3% to 53.8%. The two treatments, chronic nicotine administration and nbm lesion, also increased the size of individual neuronal responses, prolonged their duration, and shortened the response latency. Responses to glutamate were unaffected by either procedures. The results show that the increase in [3H]nicotine binding produced by chronic nicotine administration is associated with an increased response to iontophoretically applied nicotine, suggesting that the receptor upregulation induced by the chronic treatment were functional. Less easily explained is the association between increased sensitivity of frontal cortical neurons to nicotine after nbm lesion with a decreased receptor density. It is suggested that a substantial proportion of nicotinic receptors are located presynaptically, and that their loss after lesion concealed an upregulation at postsynaptic sites.

The present study investigated the performance of rats at 3-4 months and 21 months of age in the Morris water maze and correlated age-related cognitive deficits with changes in both cholinergic and GABAergic systems in the frontal cortex. The older rats were divided into two groups, unimpaired old and impaired old according to their ability to find a hidden submerged platform in the water maze, for electrophysiological, neurochemical, and morphological studies. The firing rate of frontal cortical neurones was recorded from the motor area of the frontal cortex under urethane anaesthesia and was found to be significantly slower in the two aged groups of rats compared to the young rats, but there were no differences between the two aged groups. The sensitivity of frontal cortex neurones of the impaired and unimpaired old age groups to ACh and to carbachol was significantly lower than that of the young group, but there were no differences between the two old age groups. In contrast, sensitivity of frontal cortex neurones to bicuculline was significantly higher in the aged rats compared with the young rats and was significantly greater in the impaired old rats than in the unimpaired old rats. The sensitivity of cortical neurones to glutamate was unaffected by age. There were also significant correlations between the percentages of cortical neurones responding to ACh and bicuculline and different parameters of water maze acquisition during days 7-8, but not during days 2-3, when spatial learning had not begun, and days 13-14, when spatial learning was complete. Biochemical and morphological analyses did not show any significant differences in ChAT activity and AChE-positive fibre density in the frontoparietal cortices of the three groups of rats. The results demonstrate that the learning deficit observed in old age rats cannot be adequately explained solely by a reduction in cholinergic receptor sensitivity and that an age-related increase in GABAergic tone may be a more important determinant of cognitive impairment.

One week after unilateral alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) lesions of nucleus basalis magnocellularis, rats showed significant lateralised bias in spontaneous turning and in turning induced by tail pinch or by placing the rat on a 45 degrees grid. Turning was biased to the lesioned side and this side also showed increased responsiveness to pin-prick stimulation of the skin (somaesthesia), snout and whisker stimulation and ammonia olfaction. Arecoline (0.5 mg/kg), at a dose which did not affect responses to sensorimotor stimulation in sham-operated rats, corrected the lesion-induced biased turning to tail pinch and the 45 degrees grid test and reduced the bias in the open field. In contrast, nicotine (0.05 mg/kg), at a dose which also did not substantially affect responses to sensorimotor stimulation in sham-operated rats, switched the lesion-induced turning bias towards the contralateral side. Neither cholinoceptor agonist reduced the lesion-induced increased sensory responsiveness. The effects of nicotine were blocked by the centrally acting nicotinic antagonist, mecamylamine (1.0 mg/kg), but not by hexamethonium (1.0 mg/kg), or ondansetron (0.01 mg/kg). Amphetamine (up to 1.0 mg/kg) did not affect the lesion-induced motor asymmetry. The results confirm that the basal forebrain cholinergic system plays a role in sensorimotor cortical functions, but suggest different functional roles for muscarinic and nicotinic receptors.

Unilateral lesions of the nucleus basalis magnocellularis (NBM) produced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid in rats caused, 8-10 weeks after the lesion, a 94% reduction in cortical acetylcholinesterase fibres and reduced activities of acetylcholinesterase and choline acetyltransferase by 70-80% in the frontal cortex ipsilateral to the lesion. In anaesthetized unlesioned control rats, iontophoretic administration of acetylcholine and carbachol produced atropine-sensitive inhibition and excitation of frontal cortical neurones, effects similar to those produced by electrically stimulating the NBM. The lesion reduced cortical neuronal firing rates but increased the percentage and sensitivity of neurones responding to acetylcholine, the predominant response changing from inhibition to excitation; response duration increased but latency was unaffected. The size of the response of individual neurones to carbachol, but not the percentage of sensitive neurones, was also increased in lesioned animals. The proportion of neurones responding to bicuculline and their individual sensitivities were increased by the lesion, suggesting that the lesion increased GABAergic tone; responses to glutamate were unchanged. The lesion did not affect the proportion of neurones in which acetylcholine modulated neuronal responses but reversed the nature of the modulation to predominantly excitatory; excitation was the predominant response to electrical forepaw stimulation in unlesioned control animals. This suggests a possible interaction between GABAergic and cholinergic mechanisms in selective attention and processing of cognitive information. Acute administration of di-isopropyl fluorophosphate to unlesioned animals significantly increased the number of frontal cortical neurones responding to acetylcholine, without affecting individual neuronal sensitivity or responses to carbachol and glutamate. The similarity of these effects to those of acetylcholine in lesioned animals suggests that the increased sensitivity to acetylcholine in the latter was due to loss of acetylcholinesterase, enabling diffusion of acetylcholine to more distant neurones. However, acetylcholinesterase does not hydrolyse carbachol and therefore it is necessary to postulate a different post-synaptic mechanism to explain the lesion-induced increases in the sensitivities of individual neurones to carbachol and to acetylcholine; interpretation of experimental findings should take these two mechanisms into account.

Grafts of fetal dentate gyrus (DG) and CA1 hippocampal subfield tissue were extruded into the dentate gyri of adult male Sprague-Dawley rats, 7-10 days after lesioning the granule cells with colchicine (0.06 microliter of 7 mg/ml solution at each of 5 sites/hippocampus). Graft area-host and host-graft area connectivities were investigated 4-6 months post-transplantation by recoding extracellular evoked response in hippocampal slice preparations. Following stimulation of the host mid-molecular layer, evoked field potential responses, showing considerable variation, were recorded in both types of graft. Evoked responses in the lesioned DG without grafts were recorded in very few slices. Stimulation of the area of DG tissue grafts occasionally evoked responses in the host CA3/CA4 and there was no evidence for CA1 graft area-CA3/CA4 connectivity; stimulation of DG and CA1 graft areas occasionally evoked responses in the host CA1. Responses in the area of both DG and CA1 grafts supported short-term potentiation following stimulation of the host mid-molecular layer but only DG graft areas supported long-term potentiation of the population spike amplitude. In the area of both types of transplant a tonic bicuculline-sensitive inhibition was present and paired-pulse stimulation paradigms provided some evidence for inhibition. It is possible that responses recorded within the area of grafted tissue to stimulation of the host are attributable to host-graft connectivity and similarly, responses recorded in the host to stimulation of the area of the graft may be attributable to graft-host connectivity. Only DG graft areas received host inputs which were capable of sustaining a long-term potentiation and establishing efferent contacts with the host CA3/CA4 subfield, suggesting that these would be more likely than CA1 grafts to reinstate normal functional circuitry.

Interactions between the gamma-aminobutyric acid (GABA)ergic system and the dopaminergic and cholinergic systems in the control of jaw movements, measured with a phototransducer system, were investigated in both dorsal and ventral regions of the rat striatum. Muscimol (25 and 50 ng/0.2 microliters) injected into the dorsal striatum did not affect jaw movements induced by apomorphine (0.2 mg/kg i.v.) or pilocarpine (4 mg/kg i.v.), but when injected into the ventral striatum it inhibited these jaw movements and those induced by carbachol (1 micrograms/0.2 microliters) injected into the ventral striatum. Picrotoxin (250 and 500 ng/0.2 microliters) injected into the dorsal striatum enhanced the effects of apomorphine (0.2 mg/kg) but not those of pilocarpine. When injected into the ventral striatum, picrotoxin did not affect apomorphine-induced oral movements but enhanced the effects of pilocarpine and carbachol. The benzodiazepine, flunitrazepam (100 ng/0.2 microliters), had no effect when injected in the dorsal striatum, and showed some inhibitory effects on dopaminergic and cholinergic oral movements when injected in the ventral striatum. The results suggest that the striatal GABAergic inhibitory effect on dopaminergic and cholinergic function is regionally specific, supporting a dorsal/ventral functional division of the rat striatum. The results also suggest that oral movements induced by dopaminergic and cholinergic drugs are distinct forms of repetitive oral behaviour.

True Blue, a retrograde fluorescent tracer, was used to investigate axonal outgrowth after grafting embryonic forebrain cholinergic tissue into the cholinergically-deafferented rat frontal cortex. Unilateral deafferentation was achieved by injecting alpha-amino-3-OH-4-isoxozole propionic acid (AMPA) into the ipsilateral nucleus basalis magnocellularis. Grafting occurred 3 weeks after the lesion. Eight to 10 weeks later, True Blue was injected bilaterally into the cortex at a superficial site which, on the transplanted side, was located at least 2 mm away from the transplant. Forty-eight h later, retrogradely labelled fluorescent neurones could be seen in the deepest part of the transplants. Some of these neurones were shown to be ChAt-positive, providing evidence of axonal outgrowth from transplanted cholinergic neurones into cholinergically-deafferented frontal cortex.

Nicotine, scopolamine, oxotremorine, diisopropyl-fluorophosphate (DFP) and tetrahydroaminoacridine (THA) were administered chronically to different groups of rats in doses reported to alter central muscarinic and/or nicotinic receptor numbers. Beginning 24 h after final drug injection, the groups were compared to a vehicle control group on acquisition of a hidden platform position in the Morris water maze over 20 trials with a 30-min inter-trial interval. Chronic treatment with either nicotine or scopolamine significantly improved the rate of learning, but oxotremorine and DFP retarded learning and THA had no effect on learning. The chronic drug effects on behaviour were consistent with known effects of the injected drugs on muscarinic and nicotinic binding in the forebrain and on the sensitivity of frontal cortex neurones to iontophoretically applied cholinoceptor agonists. However, alternative explanations for the observed changes cannot be ruled out, since the drugs used are known to have a wide range of effects on other neurotransmitters.

The first intron of the human Pro alpha 1(I) collagen gene contains an orientation-dependent enhancer composed of both positive and negative cis-acting elements involved in the transcriptional regulation of this gene. Deletion of a 360 bp Sau 3A intronic fragment spanning nucleotide +494 to +854 (S360) resulted in dramatic down-regulation of pCOL-KT (Thompson et al., J Biol Chem 266: 2549-2556, 1991). Using a DNaseI protection assay, we demonstrate a single footprint located at +590 to +615 in the S360 fragment; nuclear extracts prepared from mesenchymal and nonmesenchymal cells exhibited similar binding characteristics. A double stranded oligonucleotide representing a consensus Ap-1 binding sequence competed with S360 for binding. In contrast to what occurred in response to S360 deletion which was always accompanied by reduced expression, the deletion of the Ap-1 binding site (+598 to +off) caused either increased or decreased expression of the reporter gene depending on the target cell. Site-directed mutations in the Ap-1-like cis-element of Pro alpha 1(I) were also tested in transient expression assays. Consistent with the paradoxical results of Ap-1 deletion, we observed that the functional consequences of mutations in the Ap-1 site also varied in different cells. In A204 cells, one point mutation, which resulted in the loss of protein binding to S360, led to increased CAT activity while another point mutant, which retained binding of the Ap-1 like trans-acting factor(s), showed decreased CAT expression. The effects of these two mutations in the HFL-1 cells were exactly opposite of what was seen for A204 cells. Based on these observations, we postulate that the Ap-1 site plays a critical role in the transcriptional activity of the human Pro alpha 1(I) gene. The implications of an apparently dual mode of regulation through a single cis-regulatory element are discussed.

The behavioural and neuropathological effects of both systemic and intrahippocampal injections of paraquat dichloride (1,1'-dimethyl 4,4'-bipyridinium dichloride) were studied in rats. Paraquat (0.1-1.0 mumol) injected into the dorsal hippocampus, produced limbic motor seizures within a few minutes of injection followed by neuronal damage in the CA1 and CA3 pyramidal cell layers, pyriform cortex, dentate granule cell layer and in the hilus fascia dentata at 24 hr (n = 9 rats). A smaller dose of paraquat (10 nmol) was ineffective. The effects of intrahippocampal injections of paraquat (1 mumol) were prevented by administering it together with atropine (50 nmol; n = 6 rats) or by giving it 60 min. after MK 801 (0.3 mg.kg-1 intraperitoneally). Systemic injections of paraquat (20-100 mg.kg-1) also produced forelimb clonus and rearing in 10 out of 15 animals. Neuronal cell death was found 24 hr later in 9 of these rats and was restricted to the pyriform cortex, the brain region with the highest concentrations of paraquat. Atropine (150 mg.kg-1 intraperitoneally given 60 min. previously) completely prevented the motor seizures but cell death still occurred in 2 of the 6 animals tested. In conclusion, both systemic and intrahippocampal injections of paraquat produced behavioural excitation accompanied 24 hr later by brain damage and antagonist studies suggested involvement of muscarinic and NMDA receptors in the neurotoxic mechanism.

The effects of tetrahydro-9-aminoacridine (THA) were studied on the electrocortical activity of control rats and of rats with a unilateral lesion of the nucleus basalis magnocellularis (NBM) produced by alpha-amino-3-hydroxy-4-isoxozole propionic acid (AMPA). This lesion almost completely deprived the lesioned hemisphere of its cholinergic innervation. In control rats, THA (10 mg/kg i.p.) increased the amplitude of the slow components of the electrocorticogram (less than 9 Hz). These effects were antagonised by atropine (5 mg/kg i.p.). Lesion of the NBM alone decreased the amplitude of frequencies in the 12-16 Hz frequency band but did not significantly affect the slower frequencies. THA (10 mg/kg) restored the amplitude of the 12-16 Hz activity to the level seen in control rats before THA but did not affect activity in the other frequency bands. The results suggest that THA requires some residual cholinergic innervation in order to exert its effect.

The effects of bilateral intra-accumbal and intrastriatal injections of CCK-8 sulphate and its analogue, caerulein, on apomorphine-induced jaw movements were studied in ketamine-anaesthetized rats after C1 spinal transection. Jaw movements were detected by a photo-transducer attached to the mandible. CCK-8 (5, 10 and 20 ng) and its analogue, caerulein (1 and 5 ng), injected into the dorsomedial nucleus accumbens increased the frequency of apomorphine (0.2 mg/kg i.v.)-induced jaw movements. The potentiation was prevented by administration of a CCK-A receptor antagonist, lorglumide (5 ng), together with CCK-8 (20 ng) or caerulein (5 ng). Injection of lorglumide alone into the dorsomedial nucleus accumbens did not alter apomorphine (0.5 mg/kg i.v.)-induced jaw movements. Injections of CCK-8 (20 ng) and caerulein (5 ng) into adjacent sites (the ventrolateral nucleus accumbens, dorsal striatum and ventral striatum) did not affect the responses elicited by apomorphine (0.2 mg/kg i.v.). These results demonstrate that CCK-8 modulates responses elicited by a dopamine receptor agonist, apomorphine, in a region of the brain where CCK-8 is known to co-exist with dopamine.