Differential regulation of STP, LTP and LTD by structurally diverse NMDA receptor subunit-specific positive allosteric modulators.

Different types of memory are thought to rely on different types of synaptic plasticity, many of which depend on the activation of the N-Methyl-D Aspartate (NMDA) subtype of glutamate receptors. Accordingly, there is considerable interest in the possibility of using positive allosteric modulators (PAMs) of NMDA receptors (NMDARs) as cognitive enhancers. Here we firstly review the evidence that NMDA receptor-dependent forms of synaptic plasticity: short-term potentiation (STP), long-term potentiation (LTP) and long-term depression (LTD) can be pharmacologically differentiated by using NMDAR ligands. These observations suggest that PAMs of NMDAR function, depending on their subtype selectivity, might differentially regulate STP, LTP and LTD. To test this hypothesis, we secondly performed experiments in rodent hippocampal slices with UBP714 (a GluN2A/2B preferring PAM), CIQ (a GluN2C/D selective PAM) and UBP709 (a pan-PAM that potentiates all GluN2 subunits). We report here, for the first time, that: (i) UBP714 potentiates sub-maximal LTP and reduces LTD; (ii) CIQ potentiates STP without affecting LTP; (iii) UBP709 enhances LTD and decreases LTP. We conclude that PAMs can differentially regulate distinct forms of NMDAR-dependent synaptic plasticity due to their subtype selectivity.

In vivo PET imaging of the neuroinflammatory response in rat spinal cord injury using the TSPO tracer [(18)F]GE-180 and effect of docosahexaenoic acid.

PURPOSE: Traumatic spinal cord injury (SCI) is a devastating condition which affects millions of people worldwide causing major disability and substantial socioeconomic burden. There are currently no effective treatments. Modulating the neuroinflammatory (NI) response after SCI has evolved as a major therapeutic strategy. PET can be used to detect the upregulation of the 18-kDa translocator protein (TSPO), a hallmark of activated microglia in the CNS. We investigated whether PET imaging using the novel TSPO tracer [(18)F]GE-180 can be used as a clinically relevant biomarker for NI in a contusion SCI rat model, and we present data on the modulation of NI by the lipid docosahexaenoic acid (DHA). METHODS: A total of 22 adult male Wistar rats were subjected to controlled spinal cord contusion at the T10 spinal cord level. Six non-injured and ten T10 laminectomy only (LAM) animals were used as controls. A subset of six SCI animals were treated with a single intravenous dose of 250 nmol/kg DHA (SCI-DHA group) 30 min after injury; a saline-injected group of six animals was used as an injection control. PET and CT imaging was carried out 7 days after injury using the [(18)F]GE-180 radiotracer. After imaging, the animals were killed and the spinal cord dissected out for biodistribution and autoradiography studies. In vivo data were correlated with ex vivo immunohistochemistry for TSPO. RESULTS: In vivo dynamic PET imaging revealed an increase in tracer uptake in the spinal cord of the SCI animals compared with the non-injured and LAM animals from 35 min after injection (P < 0.0001; SCI vs. LAM vs. non-injured). Biodistribution and autoradiography studies confirmed the high affinity and specific [(18)F]GE-180 binding in the injured spinal cord compared with the binding in the control groups. Furthermore, they also showed decreased tracer uptake in the T10 SCI area in relation to the non-injured remainder of the spinal cord in the SCI-DHA group compared with the SCI-saline group (P < 0.05), supporting a NI modulatory effect of DHA. Immunohistochemistry showed a high level of TSPO expression (38 %) at the T10 injury site in SCI animals compared with that in the non-injured animals (6 %). CONCLUSION: [(18)F]GE-180 PET imaging can reveal areas of increased TSPO expression that can be visualized and quantified in vivo after SCI, offering a minimally invasive approach to the monitoring of NI in SCI models and providing a translatable clinical readout for the testing of new therapies.

A characterization of white matter pathology following spinal cord compression injury in the rat.

Our laboratory has previously described the characteristics of neuronal injury in a rat compression model of spinal cord injury (SCI), focussing on the impact of this injury on the gray matter. However, white matter damage is known to play a critical role in functional outcome following injury. Therefore, in the present study, we used immunohistochemistry and electron microscopy to examine the alterations to the white matter that are initiated by compression SCI applied at T12 vertebral level. A significant loss of axonal and dendritic cytoskeletal proteins was observed at the injury epicenter within 1day of injury. This was accompanied by axonal dysfunction, as demonstrated by the accumulation of ß-amyloid precursor protein (ß-APP), with a peak at 3days post-SCI. A similar, acute loss of cytoskeletal proteins was observed up to 5mm away from the injury epicenter and was particularly evident rostral to the lesion site, whereas ß-APP accumulation was prominent in tracts proximal to the injury. Early myelin loss was confirmed by myelin basic protein (MBP) immunostaining and by electron microscopy, which also highlighted the infiltration of inflammatory and red blood cells. However, 6weeks after injury, areas of new Schwann cell and oligodendrocyte myelination were observed. This study demonstrates that substantial white matter damage occurs following compression SCI in the rat. Moreover, the loss of cytoskeletal proteins and accumulation of ß-APP up to 5mm away from the lesion site within 1day of injury indicates the rapid manner in which the axonal damage extends in the rostro-caudal axis. This is likely due to both Wallerian degeneration and spread of secondary cell death, with the latter affecting axons both proximal and distal to the injury.

The effect of mechanical strain or hypoxia on cell death in subpopulations of rat dorsal root ganglion neurons in vitro.

Spinal nerves and their associated dorsal root ganglion (DRG) cells can be subject to mechanical deformation and hypoxia associated with pathology such as disc herniation, spinal stenosis and spine trauma. There is very limited information on the response of adult DRG neurons to such stressors. In this study we used an in vitro approach to examine the response of adult DRG cells to (a) mechanical, hypoxic, and combined injuries; and (b) to compare the effects on injury on nociceptive and non-nociceptive neurons, as well as on non-neuronal cells. Mechanical injury (20% tensile strain) led to significant neuronal cell death (assessed by ethidium homodimer-1 labelling), which was proportional to strain duration (5 min, 1 h, 6 h or 18 h). Hypoxia (2% O(2) for 24 h) also promoted death of DRG neurons, and was further enhanced when mechanical strain and hypoxia were combined. Both mechanical strain and hypoxia significantly decreased the maximum neurite length. Conversely, death of non-neuronal cells was only increased by hypoxia and not by mechanical strain. Total cell death in response to mechanical injury or hypoxia was similar in both non-nociceptive (neurofilament, NF-200 immunoreactive) and nociceptive (calcitonin gene-related peptide, CGRP immunoreactive) neurons, but apoptosis (assessed by activated caspase-3 immunostaining) was significantly higher in CGRP than NF-200 neurons. Surprisingly, cell death of non-peptidergic nociceptors (identified by Griffonia simplicifolia IB4 lectin binding) was already high in control cultures, and was not increased further by either mechanical stretch or hypoxia. These results provide detailed information on the response of adult DRG subpopulations to hypoxia and mechanical strain, and describe in vitro models that could be useful for screening potential neuroprotective agents.

Activation transcription factor-3 activation and the development of spinal cord degeneration in a rat model of amyotrophic lateral sclerosis.

It has been reported that an early activation of glial fibrillary acid protein (GFAP) in astroglial cells occurs simultaneously in peripheral nerves and spinal cord from the G93A SOD1 mouse model of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder. In ALS, the contribute to the pathological process of different cell types varies according to the disease stage, with a florid immune response in spinal cord at end stage disease. In this study, we have mapped in different anatomical sites the process of disease-induced functional perturbation from a pre-symptomatic stage using a marker of cellular distress expressed in neurons and glial cells, the activating transcription factor 3 (ATF-3), and applied large-scale gene expression analysis to define the pattern or transcriptional changes occurring in spinal cord from the G93A SOD1 rat model of ALS in parallel with ATF-3 neuronal activation. From the disease onset onward, transgenic lumbar spinal cord displayed ATF-3 transcriptional regulation and motor cells immunostaining in association with the over-expression of genes promoting cell growth, the functional integrity of cell organelles and involved in the modulation of immune responses. While spinal cord from the pre-symptomatic rat showed no detectable ATF-3 transcriptional regulation, ATF-3 activation was appreciated in large size neurofilament-rich, small size non-peptidergic and parvalbumin-positive neurons within the dorsal root ganglia (DRG), and in ventral roots Schwann cells alongside macrophages infiltration. This pattern of peripheral ATF-3 activation remained detectable throughout the disease process. In the G93A SOD1 rat model of ALS, signs of roots and nerves subtle distress preceded overt clinical-pathological changes, involving both glial cells and neurons that function as receptors of peripheral sensory stimuli from the muscle. In addition, factors previously described to be linked to ATF-3 activation under various experimental conditions of stress, become switched on in spinal cord from the end-stage transgenic rat model of ALS.

Neurological benefits of omega-3 fatty acids.

The central nervous system is highly enriched in long-chain polyunsaturated fatty acid (PUFA) of the omega-6 and omega-3 series. The presence of these fatty acids as structural components of neuronal membranes influences cellular function both directly, through effects on membrane properties, and also by acting as a precursor pool for lipid-derived messengers. An adequate intake of omega-3 PUFA is essential for optimal visual function and neural development. Furthermore, there is increasing evidence that increased intake of the long-chain omega-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may confer benefits in a variety of psychiatric and neurological disorders, and in particular neurodegenerative conditions. However, the mechanisms underlying these beneficial effects are still poorly understood. Recent evidence also indicates that in addition to the positive effects seen in chronic neurodegenerative conditions, omega-3 PUFA may also have significant neuroprotective potential in acute neurological injury. Thus, these compounds offer an intriguing prospect as potentially new therapeutic approaches in both chronic and acute conditions. The purpose of this article is to review the current evidence of the neurological benefits of omega-3 PUFA, looking specifically at neurodegenerative conditions and acute neurological injury.

A combination of intravenous and dietary docosahexaenoic acid significantly improves outcome after spinal cord injury.

Previous studies have shown that omega-3 polyunsaturated fatty acids such as alpha-linolenic acid and docosahexaenoic acid (DHA) are neuroprotective in models of spinal cord injury (SCI) in rodents. However, the mechanism of action underlying these effects has not been elucidated, and the optimum treatment regime remains to be defined. We have therefore carried out a detailed analysis of the effects of DHA in adult rats subject to thoracic compression SCI. Saline or DHA (250 nmol/kg) was administered intravenously (i.v.) 30 min after compression. After injury, the saline group received a standard control diet for 1 or 6 weeks, whereas DHA-injected animals received either a control or a DHA-enriched diet (400 mg/kg/day) for 1 or 6 weeks. Other groups received a DHA-enriched diet only for 1 week following injury, or received acute DHA (250 nmol/kg; i.v.) treatment delayed up to 3 h after injury. We also assessed oxidative stress and the inflammatory reaction at the injury site, neuronal and oligodendrocyte survival and axonal damage and the locomotor recovery. At 24 h, lipid peroxidation, protein oxidation, RNA/DNA oxidation and the induction of cyclooxygenase-2 were all significantly reduced by i.v. DHA administration. At 1 week and 6 weeks, macrophage recruitment was reduced and neuronal and oligodendrocyte survival was substantially increased. Axonal injury was reduced at 6 weeks. Locomotor recovery was improved from day 4, and sustained up to 6 weeks. Rats treated with a DHA-enriched diet in addition to the acute DHA injection were not significantly different from the acute DHA-treated animals at 1 week, but at 6 weeks showed additional improvements in both functional and histological outcomes. DHA treatment was ineffective if the acute injection was delayed until 3 h post-injury, or if the DHA was administered for 1 week solely by diet. Our results in a clinically relevant model of SCI show that significant neuroprotection can be obtained by combining an initial acute i.v. injection of DHA with a sustained dietary supplementation. Given that the safety and tolerability of preparations enriched in omega-3 fatty acids is already well-documented, such a combined DHA treatment regime deserves consideration as a very promising approach to SCI management.

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat.

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 microg/kg) or CEPO (40 microg/kg). Results indicated that 3 days post-injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase-mediated dUTP nick-end labelling (TUNEL)-labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker beta-amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the beta common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.

Dietary enrichment with omega-3 polyunsaturated fatty acids reverses age-related decreases in the GluR2 and NR2B glutamate receptor subunits in rat forebrain.

Ageing is associated with a decrease in the brain content of omega-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and with decreased neuroplasticity. The glutamate receptor subunits GluR2 and NR2B play a significant role in forebrain synaptic plasticity. We investigated GluR2 and NR2B in the aged prefrontal cortex, hippocampus and striatum, and tested if treatment with a preparation containing EPA and DHA can reverse age-related changes. The study compared adult and old (3-4 and 24-26 month) rats, and the latter were fed a standard diet or a diet supplemented for 12 weeks with omega-3 PUFA at 270mg/kg/day (ratio EPA to DHA 1.5:1). Ageing was associated with decreases in the GluR2 and NR2B subunits in all structures. These decreases were fully reversed by omega-3 PUFA supplementation. Age-related changes in the phospholipid PUFA content were also seen. Decreases in DHA were mostly corrected by supplementation. This study supports the neuroprotective effect of omega-3 fatty acids in brain ageing, and illustrates specific mechanisms underlying this effect.

In vivo metabolism and clearance of substance P and co-expressed tachykinins in rat striatum.

Neurons expressing the preprotachykinin A gene, which encodes the sequences of substance P, neurokinin A, neuropeptide gamma and neuropeptide K, exemplify peptide co-existence. Furthermore, there is also evidence that substance P fragments have biological activity. However, the relative contribution of each of these peptides to tachykinin signalling is still poorly understood. An important factor which will determine the characteristics of the signal mediated by co-localised peptides is their clearance from the extracellular space. The striatum, in which tachykinins are present and exert neuromodulatory roles, can be used as a model to investigate this aspect. Therefore, in this study we characterised in vivo in the striatum the metabolism and clearance of substance P and of the other three co-expressed peptides. After intrastriatal administration of 1 pmol, tritiated substance P disappeared too rapidly for metabolites to be detected. However, when 10 nmol substance P and 1 pmol tritiated substance P were co-injected, substance P(1-4) and substance P(1-7), which are biologically active, were detected as major metabolites. Under these conditions, the rate of decay of tritiated substance P was 0.2 nmol/min. The effects of the peptidase inhibitors thiorphan, bestatin and captopril suggested that neutral endopeptidase 24.11 and aminopeptidases were involved in primary substance P cleavages, whereas angiotensin-converting enzyme was involved in secondary cleavages. The monitoring of the decay of unlabelled substance P by high-performance liquid chromatography gave a rate of 0.16 nmol/min. Using high-performance liquid chromatography with capillary electrophoresis, the rates of decay of 10 nmol neurokinin A or neuropeptide gamma were five and seven times faster than that of substance P. In contrast, over the time course of the experiment, no significant decay of neuropeptide K was detected. These results show that substance P disappears rapidly from the extracellular space, and supports the formation in vivo of major N-terminal active substance P metabolites. Our study also highlights significant differences in the clearance of co-expressed tachykinins and suggests that certain species may disappear relatively slowly from the extracellular space, and thus may make a significant temporal and spatial contribution to signalling.

Effects of sevoflurane on dopamine, glutamate and aspartate release in an in vitro model of cerebral ischaemia.

Release of excitatory amino acids and dopamine plays a central role in neuronal damage after cerebral ischaemia. In the present study, we used an in vitro model of ischaemia to investigate the effects of sevoflurane on dopamine, glutamate and aspartate efflux from rat corticostriatal slices. Slices were superfused with artificial cerebrospinal fluid at 34 degrees C and episodes of 'ischaemia' were mimicked by removal of oxygen and reduction in glucose concentration from 4 to 2 mmol litre(-1) for < or = 30 min. Dopamine efflux was monitored in situ by voltammetry while glutamate and aspartate concentrations in samples of the superfusate were measured by HPLC with fluorescence detection. Neurotransmitter outflow from slices was measured in the absence or presence of sevoflurane (4%). After induction of ischaemia in control slices, there was a mean (SEM) delay of 166 (7) s (n = 5) before sudden efflux of dopamine which reached a maximum extracellular concentration of 77.0 (15.2) micromol litre(-1). Sevoflurane (4%) reduced the rate of dopamine efflux during ischaemia (6.90 (1.5) and 4.73 (1.76) micromol litre(-1) s(-1) in controls and sevoflurane-treated slices, respectively; P<0.05), without affecting its onset or magnitude. Excitatory amino acid efflux was much slower. lschaemia-induced glutamate efflux had not reached maximum after 30 min of ischaemia. Basal (pre-ischaemic) glutamate and aspartate efflux per slice was 94.8 (24.8) and 69.3 (31.5) nmol litre(-1) superfusate (n = 4) and was not significantly reduced by 4% sevoflurane. lschaemia greatly increased glutamate and aspartate efflux (to a maximum of 919 (244)% and 974 (489)% of control, respectively). However, ischaemia-induced efflux of both glutamate and aspartate was significantly reduced by 4% sevoflurane (P < 0.001 for glutamate, P < 0.01 for aspartate). In summary, sevoflurane may owe part of its reported neuroprotective effect to a reduction of ischaemia-induced efflux of excitatory amino acids and, to a lesser extent, dopamine.

Substance P modulation of striatal dopamine outflow is determined by M(1) and M(2) muscarinic receptors in male wistar rats.

Substance P (SP) stimulates striatal dopamine outflow through a cholinergic muscarinic link. SP-induced increase in acetylcholine (Ach) is concentration-dependent, whereas the stimulation of dopamine outflow is seen only over a limited concentration range. M(1) and M(2) receptor stimulation has opposite effects on dopamine outflow. We postulated that the effect of SP on dopamine outflow depends on the M(1)/M(2) balance. We show that Ach (10-2500 microM) stimulates dopamine outflow in striatal slices in a biphasic manner, similar to SP (0.01-100 nM). An inactive SP concentration (10 nM) which was higher than the active concentration range, became active in the presence of the M(2) antagonist methoctramine (100 microM). Conversely, the effect of 1 nM SP was reversed by the M(1) antagonist pirenzepine (1 microM). Our observations show that SP modulation of dopamine outflow is determined by a balance between M(1) and M(2) receptors.

Imipramine and phenelzine decrease glutamate overflow in the prefrontal cortex--a possible mechanism of neuroprotection in major depression?

Antidepressant drugs have been used for decades, but the neurobiological substrate of their efficacy is not completely understood. Although these drugs have well-established effects on monoamines, evidence is emerging that they may also affect other neurotransmitter systems. It has been shown that treatment with a wide range of antidepressants changes the binding characteristics of the N-methyl-D-aspartate type of glutamate receptor. This change is delayed and occurs only in the cortex. The mechanism that triggers it is unknown. We hypothesized that N-methyl-D-aspartate receptor alterations may be due to changes in the dynamics of cortical excitatory amino acid release. Such changes are of particular interest in areas such as the prefrontal cortex, a region involved in stress responses and affected in major depression. We investigated the effects of two antidepressants with different modes of action, imipramine and phenelzine, on glutamate and aspartate outflow in rat prefrontal cortex and striatum. We showed that antidepressants significantly decreased stimulated glutamate outflow. The effect had a rapid onset, was sustained during chronic administration and was only seen in the prefrontal cortex. This change may initiate receptor alterations. Furthermore, if antidepressants can dampen states of hyperglutamatergic activity and the subsequent excitotoxicity, their chronic use may have a considerable neuroprotective potential in major depression.

N- and C-terminal substance P fragments: differential effects on striatal [3H]substance P binding and NK1 receptor internalization.

N- and C-terminal substance P (SP) fragments increase striatal dopamine outflow at nanomolar concentrations. This contrasts with their low affinity for NK1 receptors. To explore this discrepancy, we investigated the interaction of SP and SP fragments with NK1 sites in fresh striatal slices, the same model used in the functional studies on dopamine outflow. [3H]SP bound specifically to one site (Kd = 6.6 +/- 0.9 nM; Bmax = 12.6 +/- 0.7 fmol/mg protein). [3H]SP binding was displaced by SP (IC50 = 11.8 nM), but not by SP(1-7) or SP(5-11), up to 10 microM. In contrast, 10 nM SP(1-7) or SP(5-11) induced significant internalization of the NK1 receptor, similar to that induced by SP. We suggest that SP fragments have high affinity for an NK1 receptor conformer which is different from that labelled by [3H]SP.

Identification of substance P metabolites using a combination of reversed-phase high-performance liquid chromatography and capillary electrophoresis.

Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1-4) and SP(1-7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide.

Substance P fragments and striatal endogenous dopamine outflow: interaction with substance P.

Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1-9) and substance P(6-11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1-9) and substance P-(6-11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6-11) (1 nM) were reversed by the tachykinin NK1 antagonist WIN 51,708 (17beta-hydroxy-17alpha-ethynyl-5alpha-androstano[3,2- b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1-9) were not modified by the antagonist. Substance P-(1-9) and substance P-(6-11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCI. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 microM) but not by nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11). The data show that substance P-(1-9) and substance P-(6-11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.

N- and C-terminal substance P fragments modulate striatal dopamine outflow through a cholinergic link mediated by muscarinic receptors.

The present study investigated whether the modulatory effects of substance P and substance P fragments on striatal dopamine release involve a cholinergic link. Rat striatal slices were incubated with substance P, substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11) in the absence or presence of various agents which modify cholinergic transmissions, and endogenous dopamine outflow was measured using high-performance liquid chromatography. The incubation of striatal slices with substance P and its N- and C-terminal fragments (1 nM) induced a significant overflow of endogenous dopamine. Neostigmine (150 nM) potentiated the effects of substance P and its fragments, whereas the incubation with hemicholinium-3 (50 microM) abolished the effects of the peptides on dopamine outflow. The acetylcholinesterase inhibitor and the inhibitor of choline uptake did not have intrinsic effects on dopamine outflow. The muscarinic antagonist atropine (1 microM) reversed completely the effects of substance P and its fragments, whereas the nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM) were devoid of effects. None of the cholinergic antagonists modified dopamine outflow. The results suggest that substance P and several N- and C-terminal substance P fragments activate cholinergic neurons in striatal slices. The released acetylcholine induces an increased dopamine outflow, mediated by muscarinic receptors. These observations represent additional evidence which supports the functional interactions between substance P, acetylcholine and dopamine in the striatum. Furthermore, they show that substance P fragments may exert neuromodulatory effects through mechanisms similar to those underlying the effects of the parent peptide.

Distribution and excretion of radiolabeled temoporfin in a murine tumor model.

The biodistribution and excretion of temoporfin (tetra[m-hydroxyphenyl]chlorin, m-THPC), a recently developed photosensitizer, was investigated in BALB/c mice. [14C]temoporfin was administered intravenously (0.73 mumol/kg) to tumor-free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood, tissue and fecal samples were collected for 35 days and 10 days postdose from tumor-free mice and tumor-bearing mice, respectively. Blood concentrations fell rapidly such that at later time points they were indistinguishable from background counts. Tumor concentrations rose to a peak of 0.34 microgram temoporfin equivalents/mL at 2 days and then declined in parallel (log plot) with the blood concentrations. Tumor: tissue ratios at 2 days for skin, adipose tissue and skeletal muscle underlying the tumor were 1.5, 2.3 and 3.8, respectively. By 4 days the corresponding values were 1.6, 3.4 and 4.0. Nearly 40% of the administered radioactivity was excreted in the feces in the first 24 h and more than 80% had been excreted by 20 days. Less than 0.2% of the dose was recovered from the urine. An elimination half-life of 10-12 days was calculated from the excretion data.

Evidence for modulatory effects of substance P fragments (1-4) and (8-11) on endogenous dopamine outflow in rat striatal slices.

The effects of substance P-(1-4) and substance P-(8-11) on endogenous dopamine outflow in rat striatal slices were investigated. The dose-response curves (0.01 nM to 1 microM) were bell-shaped for both peptides, with significant increases in dopamine outflow at 0.1 and 1 nM. Dopamine overflow elicited by 1 nM substance P-(1-4) or substance P-(8-11) and 25 mM KCl was additive. Although substance P-(8-11) contains a truncated tachykinin sequence, the tachykinin NK1 receptor antagonist WIN 51,708 (17 beta-hydroxy-17 alpha-ethynyl-5 alpha-androstano[3,2-b]pyrimido[1,2- a]benzimidazole (2.5 nM) fully reversed its effect. The interaction between the antagonist and 1 nM substance P-(1-4) was statistically not significant. The data constitute the first evidence that the fragments substance P-(1-4) and substance P-(8-11) could exert central effects and suggest that they may play a role in neuromodulation in the basal ganglia.

Binding of temoporfin to the lipoprotein fractions of human serum.

The binding of a new photosensitizer, temoporfin, to human serum lipoproteins was investigated. [14C]-Temoporfin (0.1-10 micrograms ml-1) was incubated with human serum for 30 min at room temperature or for 20 h at 4 degrees C, prior to stepwise density flotation to separate the lipoprotein fractions. The distribution of the drug was independent of the initial concentration or time and temperature of the incubation. The proportion of temoporfin in each fraction was: very low density lipoprotein 6%, low density lipoprotein 22%, lipoprotein(a) 17%, high density lipoprotein 39% and lipoprotein deficient serum 16%. Autoradiography of agarose gels showed that the drug was associated with the lipoprotein in the fractions. Fractionation of plasma samples collected from a patient after an intravenous infusion of temoporfin revealed a binding profile similar to that obtained in the in vitro study.