Conditioned appetitive stimulus increases extracellular dopamine in the nucleus accumbens of the rat.

This study used in vivo microdialysis to examine the release of dopamine (DA) in the nucleus accumbens (nAc) during the performance of a previously learned, signalled sucrose reward task, and during conditioning of a neutral tone stimulus to this reward. Behavioural measures (magazine entries) confirmed that stimuli associated with sucrose presentation became secondary rewarding stimuli, and DA release was also monitored during subsequent presentation of these stimuli alone. Perhaps surprisingly, during magazine entry for consumption of sucrose, i.e. in conditions similar to routine training, dialysate DA levels in the nAc did not increase. In contrast, during conditioning of the tone with light-sucrose, dopamine levels increased consistently and significantly. Interestingly, DA levels were somewhat, but significantly, increased when tone alone was presented in a test session, i.e. two hours after conditioning, and even more so when tone was combined with the light previously associated with sucrose. In this latter case the number of magazine entries increased to a level similar to that seen during conditioning. Presentation of light alone resulted in a similar level of magazine entries to tone alone, but no significant increase in DA. In summary, these studies confirm that a neutral stimulus can acquire the behavioural properties of reward when conditioned. The neurochemical data, on the other hand, suggest that increases of DA in nAc are more likely to be related to new associative learning than to established incentive or consumatory processes. The increase in DA release in the test session may be related either to the secondary reinforcing properties acquired by the stimulus, or to the change in contingencies, or to the aversive effects of the omission of reward.

Modulatory effects of L-DOPA on D2 dopamine receptors in rat striatum, measured using in vivo microdialysis and PET.

Putative modulatory effects of L-3,4-dihydroxyphenylalanine (L-DOPA) on D2 dopamine receptor function in the striatum of anaesthetised rats were investigated using both in vivo microdialysis and positron emission tomography (PET) with carbon-11 labelled raclopride as a selective D2 receptor ligand. A single dose of L-DOPA (20 or 100mg/kg i.p.) resulted in an increase in [11C]raclopride binding potential which was also observed in the presence of the central aromatic decarboxylase inhibitor NSD 1015, confirming that the effect was independent of dopamine. This L-DOPA evoked D2 receptor sensitisation was abolished by a prior, long-term administration of L-DOPA in drinking water (5 weeks, 170mg/kg/day). In the course of acute L-DOPA treatment (20mg/kg), extracellular GABA levels were reduced by approximately 20% in the globus pallidus. It is likely that L-DOPA sensitising effect on striatal D2 receptors, as confirmed by PET, may implicate striato-pallidal neurones, hence a reduced GABA-ergic output in the projection area. Since the L-DOPA evoked striatal D2 receptor supersensitivity habituates during long-term treatment, the effects reported here may contribute to the fluctuations observed during chronic L-DOPA therapy in Parkinson's disease.

Increased extracellular dopamine in the nucleus accumbens of the rat during associative learning of neutral stimuli.

Brain microdialysis was used to study changes in dopamine in the nucleus accumbens and the dorsal striatum during associative learning between two neutral stimuli, flashing light and tone, presented on a paired schedule during stage 1 of a sensory preconditioning paradigm. The tone was subsequently paired with mild footshock using standard aversive conditioning procedures and the formation of a conditioned association between the flashing light and the tone in stage 1 was assessed by measuring the ability of the flashing light to elicit the same conditioned response as the tone when presented at test. The first experiment used behavioural monitoring only, to establish stimulus parameters for subsequent microdialysis experiments. Animals receiving paired presentation of the light and tone in stage 1 showed a conditioned suppression of licking to the light as well as to the tone, indicating that associative learning between the flashing light and the tone had occurred during stage 1, whilst in a separate group of animals given the same stimuli over the same time period but on an explicitly non-paired schedule, the conditioned emotional response was seen to the tone, but not to the light, showing that no association had been formed between the two stimuli during stage 1. In dialysis experiments using the same procedure, we measured a two-fold rise in dopamine in the nucleus accumbens during paired presentation of flashing light and tone, but not during non-paired presentation of the two stimuli. On subsequent test presentation of the two stimuli, we saw increases in accumbal dopamine on presentation of the tone in both groups, reflecting the formation of an association with the footshock in both. However the flashing light elicited an increase in dopamine only in the group which had received paired presentation at stage 1. Thus accumbal dopamine release at test is correlated to the ability of the stimulus to evoke a conditioned response measured behaviourally. Hypotheses of the behavioural function of the mesolimbic dopamine system centre on its role in mediating the effects of biological reinforcers, both rewarding and aversive, conditioned and unconditioned. The present results, showing increases in extracellular dopamine in the nucleus accumbens when an association is formed between two stimuli of which neither is a biological reinforcer nor, prior to formation of the association, affects dopamine levels, suggest a role for accumbal dopamine in the modulation of associative learning in general, not only that involving reinforcement.

Effect of L-dopa and 6-hydroxydopamine lesioning on [11C]raclopride binding in rat striatum, quantified using PET.

A positron emission tomograph (PET) was used to image D2 dopamine receptor function in rat striata and to obtain regional time-radioactivity curves from individual rat brains following i.v. injection of carbon-11-labelled raclopride. Despite the limited resolution of the camera, together with associated spillover and partial volume effects, the kinetic data obtained from striata were such that specific binding of the radioligand could be quantified unilaterally, using a reference tissue compartmental model, with cerebellum data as an indirect input function. With the exception that the rat is anaesthetised, the experimental system is analogous to the acquisition and collection of clinical PET data and, by using animal models of disease, can be used to aid the interpretation of clinical studies. Using 6-hydroxydopamine (6-OHDA) lesioning of the substantia nigra pars compacta to produce a rat hemiparkinsonian model, the present results confirm that deafferentation causes a supersensitivity of post-synaptic D2 dopamine receptors. Saturation studies indicated that the measured 23% increase in [11C]raclopride binding potential reflected a change in receptor affinity. Modulation of extracellular dopamine concentration, monitored by in vivo microdialysis, demonstrated that the increased binding was unlikely to be due to a reduction in receptor occupancy by endogenous dopamine. Acute administration of L-3,4-dihydroxyphenylalanine (L-dopa) also caused an increase in [11C]raclopride binding potential, confirming the suggestion that L-dopa plays a more complex role than that of dopamine precursor in the nigrostriatal pathway.

Evaluation of [O-methyl-3H]WAY-100635 as an in vivo radioligand for 5-HT1A receptors in rat brain.

N-(2-(4-(2-Methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) is a new, potent and selective 5-HT1A receptor antagonist. We have evaluated radiolabelled WAY-100635 as a prospective radioligand for positron emission tomography (PET) by studying biodistribution in rat ex vivo. After intravenous injection, [O-methyl-3H]WAY-100635 cleared rapidly from plasma but was retained in brain. Specific binding was quantified from kinetic studies, using a reference-tissue compartment model, fitting for binding potential (k3/k4). The regional variation in binding potential correlated with the known distribution of 5-HT1A receptors. Saturation studies gave Bmax values in vivo that were consistent with those reported in vitro. At 60 min after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions (e.g. septum, entorhinal cortex and hippocampus) to that in cerebellum reached approximately 16. Pre-dosing the rats with WAY-100635 (2 mg/kg) reduced this ratio to one, whereas similar pre-dosing with citalopram (5-HT uptake site inhibitor), prazosin (alpha 1A-adrenoceptor antagonist) or idazoxan (alpha 2-adrenoceptor antagonist) caused little or no reduction. Substantial (77%) blockade of [3H]WAY-100635 binding was achieved with the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the partial agonists, ipsapirone and buspirone. Thus, the properties of WAY-100635 are such that, when labelled with carbon-11, it could provide a radioligand suitable for clinical and pharmacological investigations of central 5-HT1A receptors in man using PET.

Magnetic resonance imaging of propagating waves of spreading depression in the anaesthetised rat.

Gradient echo magnetic resonance (MR) imaging was used to demonstrate propagating waves of cortical spreading depression (SD) in the anaesthetised rat. SD was initiated by remote perfusion with 150 mM KCl applied for 0.5-2 min to the left parietal cortex. Gradient echo MR images were obtained every 12-30 s in either a vertical coronal section or a horizontal section including the superficial cortex in plan view. Within 2 min of application of KCl, we observed a zone of increased signal intensity (3-15%) on the MR image, up to 2 mm across, lasting approximately 1 min and propagating away from the site of initiation. The mean velocity for 27 of such waves seen in seven animals was calculated to be 2.79 mm/min, with means (+/- SD) in individual animals averaging 2.90 +/- 0.46 mm/min (n = 7). Increased signal intensity in gradient echo images has been attributed to an increased level of oxygenation within the venous blood. Our results are consistent with this interpretation although other physiological changes during SD may also contribute to the signal changes.

Quantification of in vivo binding of [3H]RX 821002 in rat brain: evaluation as a radioligand for central alpha 2-adrenoceptors.

On the basis of its established in vitro characteristics, [3H]RX 821002 was evaluated in rats as an in vivo radioligand for central alpha 2-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the alpha 2-antagonists, idazoxan or yohimbine, the alpha 2-agonist, clonidine, or the alpha 1-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [3H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [3H]RX 821002 can be used experimentally as an in vivo marker for central alpha 2-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [11C]RX 821002 for clinical PET studies.

Quantitation of carbon-11-labeled raclopride in rat striatum using positron emission tomography.

Using conventional autoradiographic and tissue counting techniques, the experimental quantitation of in vivo kinetics of prospective or established radioligands for PET is animal and labour intensive. The present study tested the feasibility of using PET itself to quantitate the specific binding of [11C]raclopride to rat striatum and to study the effects of experimental manipulation of endogenous dopamine on binding parameters. Carbon-11-labeled raclopride was given i.v. to anaesthetised rats, positioned in a PET camera and dynamic emission scans acquired over 60 min. Time-activity curves were generated for selected regions of interest, representing striatum and cerebellum and the striatal data fitted to a compartmental model, using cerebellum as the input function, thus circumventing the need for individual metabolite-corrected plasma curves. In control rats, the binding potential (BP), defined as the ratio of the rate constants for transfer from "free to bound" and "bound to free" compartments, was of the order of 0.6. This was reduced threefold by predosing with nonradioactive raclopride. Increasing extracellular dopamine levels by predosing with d-amphetamine resulted in a significant decrease in BP whereas reducing extracellular dopamine by predosing with gamma-butyrolactone caused a significant increase. Thus, despite the limitation in spatial resolution of PET, specific binding of raclopride could be assessed from regional time-activity curves from individual rats. The system was sufficiently sensitive that changes in BP could be detected following modulation of endogenous dopamine levels, a finding of potential relevance to the interpretation of clinical PET data.

Diffusion-weighted imaging of kainic acid lesions in the rat brain.

We present T2-weighted and diffusion-weighted images of kainic acid lesions in the rat brain. Our observations show improved image contrast between edematous lesions and unaffected tissue using diffusion-weighted imaging. Furthermore, we show that the anisotropic intensity changes associated with this sequence can be used to highlight white matter tracts and to provide information concerning their orientation in the rat brain.

Citalopram: labelling with carbon-11 and evaluation in rat as a potential radioligand for in vivo PET studies of 5-HT re-uptake sites.

In vivo autoradiography of [N-methyl-3H]citalopram in rat brain shows a differential regional localization which correlates with the localization of 5-HT re-uptake binding sites defined in vitro. A comparison of the biodistribution of [N-methyl-3H]citalopram over 2 h after i.v. injection in (1) control rats (2) rats pre-dosed with either citalopram or paroxetine and (3) rats chemically-lesioned with p-chloroamphetamine provides an estimate of specific binding relative to total binding in vivo. The ratio of binding in certain regions (e.g. cingulate) to binding in a reference tissue (e.g. cerebellum) at 30-120 min post injection is c. 1.4. In view of these results a method was developed for labelling citalopram with carbon-11 (t1/2 = 20.3 min, beta + = 99.8%) to provide a potential radioligand for studies using positron emission tomography. Thus, reaction of nca [11C]iodomethane, prepared from cyclotron-produced [11C]carbon dioxide, with norcitalopram in ethanol containing 2,2,6,6-tetramethyl-piperidine for 5 min at 95 degrees C gives crude [N-methyl-11C]citalopram in 60% radiochemical yield, decay-corrected. HPLC on silica gel provides radiochemically and chemically pure [N-methyl-11C]citalopram, as assessed by TLC, HPLC and MS. This product (isolated radiochemical yield, 49%) is easily formulated for i.v. injection. Up to 2 GBq of formulated product with a specific activity of c. 15 GBq/mumol have been prepared within 40 min from the end of radionuclide production. The described radiosynthesis has also been applied to give the single biologically active (+)-enantiomer of [N-methyl-11C]citalopram rather than the racemate. This product gives enhanced specific signal in the rat following i.v. injection, the ratio of uptake in regions of interest relative to cerebellum approaching 2 at 90 min.

Compartmental analysis of diprenorphine binding to opiate receptors in the rat in vivo and its comparison with equilibrium data in vitro.

The regional binding of the opiate receptor ligand diprenorphine has been examined in rat brain both in vivo and in vitro. The time course of total label in specific brain regions was followed up to 2 h after intravenous bolus injection of [3H]diprenorphine, with or without a pulse chase of unlabelled diprenorphine at 30 min. In addition, total label was measured 30 min after injection of labelled diprenorphine at nontracer concentrations over a range of specific activities. Total data sets for each region were fitted simultaneously to a compartmental model to give estimates of maximal binding capacity (Bmax), the second-order apparent association rate constant, and the first-order dissociation rate constant of the receptor-ligand complex. The model incorporated the use of a reference region with low specific binding (cerebellum). The binding of diprenorphine to rat brain homogenates was measured in vitro under equilibrium conditions at 37 degrees C, pH 7.4, in the presence and absence of naloxone, to give corresponding regional estimates of Bmax and the half-saturation constant Kd. The results showed a close correlation between in vitro and in vivo regional estimates of Bmax over a wide range. There were no significant interregional differences either in Kd in vitro or in the Kd derived from the in vivo analysis, although in vitro and in vivo estimates differed by an order of magnitude. This work was carried out as part of a validation study with a view to the application of the compartmental model to data obtained in vivo in humans using positron emission tomography, when successive studies over a range of specific activities are not feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo binding to peripheral benzodiazepine binding sites in lesioned rat brain: comparison between [3H]PK11195 and [18F]PK14105 as markers for neuronal damage.

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.

The radiosynthesis of [18F]PK 14105 as an alternative radioligand for peripheral type benzodiazepine binding sites.

A method has been developed for labelling PK 14105 [N-methyl-N-(1-methyl-propyl)-1(2-fluoro-5-nitrophenyl)isoquinoline-3- carboxamide], a ligand that has high affinity and selectivity for peripheral type benzodiazepine binding sites (PBBS), with NCA fluorine-18 (t1/2 = 109.8 min, beta + = 96.9%). The method involves treating the 2-chloro-analogue with cyclotron-produced NCA [18F]fluoride in dimethyl sulphoxide, with rubidium carbonate as base, at 140 degrees C for 20 min. Purification is achieved by separation on a reverse phase Sep-Pak followed by PHLC on a silica gel column, to give chemically and radiochemically pure product with a specific activity of ca 7.4 GBq/mumol (200 mCi/mumol), decay-corrected to the end of radionuclide production (EOB). The radiosynthesis requires 210 min. giving a radiochemical yield of 10-20%, decay-corrected to EOB. [18F]PK 14105 was found to bind avidly to sites associated with kainic acid-induced unilateral lesions of rat striata. Such binding was blocked by pre-dosing the rat with PK 11195, so providing evidence for specific binding to PBBS. These results suggest that [18F]PK 14105 has potential for studying phenomena associated with PBBS in man by PET.

Vascular and epithelial damage in the lung of the mouse after X rays or neutrons.

The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year. Furthermore, a dose which had no effect on leakage caused a marked increase in collagen biosynthesis. Thus the present results do not support a causal relationship between exudation of vascular protein during pneumonitis and the later development of fibrosis.

In vivo labelling of the NMDA receptor channel complex by [3H]MK-801.

An in vivo radioligand binding assay for the N-methyl-D-aspartate (NMDA) receptor channel complex in the mouse brain has been developed using the non-competitive NMDA receptor antagonist [3H]MK-801. In vivo binding of [3H]MK-801 was displaced by MK-801 (ED50 = 0.17 mg/kg i.p.), (-)-MK-801 (1.0 mg/kg), thienylcyclohexylpiperidine (1.8 mg/kg), etoxadrol (5.1 mg/kg) and (+)-SKF 10,047 (34.5 mg/kg). The potency of these drugs in this in vivo binding assay was highly correlated (r = 0.97) with their functional effects as antagonists of N-methyl-DL-aspartate-induced tonic convulsions.

Thermotolerance induced by fractionated hyperthermia: dependence of the interval between fractions.

The induction of thermotolerance by fractionated hyperthermia was investigated in the mouse ear. Ears were heated at 43.5 degrees C by immersion in water. One to ten treatments of 20 min were followed by test treatments. Thermotolerance was assessed as the increase in the duration of the test treatment required for a thermal response in 50 per cent of the ears (NT50). A single treatment induced thermotolerance which reached a maximum at 24 h when the NT50 was increased by a factor of 2.4. The same maximum was observed after each fractionated treatment used in the present study. The time course of development, however, depended on the interval between fractions. (1) When the interval was too short to allow development of thermotolerance after a single fraction (4 h), thermotolerance was not induced during fractionated treatment but it developed during the first 24 h after treatment. (2) When the interval between fractions allowed the maximal development of thermotolerance (24 h), this maximum was maintained during fractionated treatment and persisted for 24 h after treatment. (3) When the interval allowed some decay of thermotolerance (72 or 168 h) there was a further increase to maximal thermotolerance after each fraction. The decay of thermotolerance from the maximum did not depend on the interval between fractions. These results indicate that the degree of thermotolerance may fluctuate during fractionated hyperthermia.

A long-term effect of prior irradiation on the thermal enhancement of radiation damage in the mouse ear.

The responses of the mouse ear to heat alone, X-rays alone or X-rays combined with heat were measured at 10 months after initial X-ray treatments (19 Gy or 10 X 3.8 Gy), which caused similar acute reactions. Fractionating the initial dose had little effect on the response to retreatment. Prior irradiation increased thermal sensitivity so that the heating time at 43.5 degrees C required to cause necrosis was about 65 per cent that in age-matched controls. Prior irradiation also increased the response to X-rays alone, but had different effects on the susceptibilities to develop acute radiodermatitis and late deformity. For acute radiodermatitis, the second X-ray dose required to cause a given response in previously irradiated ears was 80-90 per cent that in age-matched controls and for late deformity it was 60-65 per cent. Prior irradiation had the same effects on the responses to X-rays given 6 min before mild hyperthermia (43.5 degrees C, 12 min) as on those to X-rays alone but had little effect on the responses to X-rays given 6 min after hyperthermia. Consequently, the thermal enhancement ratios for heat given after X-rays did not depend on prior irradiation whereas those for heat given before X-rays were reduced. This reduction may be due to a reduced ability of irradiated blood vessels to elicit an hyperaemic response to heat.

Altered turnover and synthesis rates of lung surfactant following thoracic irradiation.

Between 2-6 weeks after thoracic irradiation with 10 Gy X rays, when levels of surfactant in the alveoli show the greatest increase, there is a reduction in the rate of radioactivity loss from 3H-choline labeled disaturated phosphatidylcholine from the lung. This indicates a reduced turnover of surfactant. Discrepancies between the halving times for specific activity and for total radioactivity of the disaturated phospholipids suggest that at between 2 and 3 weeks post-irradiation, removal and degradation of surfactant almost ceases, but that synthesis continues normally. However, by 3 weeks post-irradiation, choline-3H incorporation into disaturated phosphatidylcholine suggests that surfactant synthesis is increased about two-fold. The reduced number of macrophages recovered from alveolar lavage between about 2 and 6 weeks post-irradiation may indicate a reason for the lengthened turnover times of surfactant over this period. Nevertheless the stimulated surfactant production that takes place from about 3 weeks onward suggests an additional active response to radiation or to radiation damage by the type II pneumonocytes. These studies confirm that the maximum levels of alveolar surfactant seen at 3 weeks post-irradiation result from a different lung response than that responsible for the increase in surfactant, which occurs within hours of irradiation.