Neutralization of the chemokine CXCL10 enhances tissue sparing and angiogenesis following spinal cord injury

After spinal cord injury, there is a chemoattractant-mediated inflammatory response that is associated with secondary degeneration. The chemoattractant CXCL10 recruits CD4 Th1 cells via the CXCR3A receptor and inhibits growth and chemotaxis of endothelial cells via the CXCR3B receptor. To test the hypothesis that CXCL10 inhibits angiogenesis following spinal cord injury, we assayed the brainstems and spinal cords of spinal cord-injured mice treated with anti-CXCL10 antibodies for expression of angiogenesis-associated genes and quantified blood vessels within their spinal cords. Brainstem microarray analysis indicated eight angiogenesis-associated genes that had significantly higher expression levels in the treated mice than in the untreated mice. Ribonuclease protection assays of the spinal cords showed a significant increase in eight angiogenesis-associated genes in treated animals compared with untreated animals. Histological analysis of the spinal cords of treated and untreated mice showed a significant increase in the number of blood vessels in treated animals. We conclude that CXCL10 plays a critical role in vasculature remodeling following spinal cord injury and that angiogenesis is enhanced following anti-CXCL10 treatment of spinal cord injuries. Improved blood flow and oxygen supply to the injury site may contribute to the functional improvement associated with this treatment.     

Immunocytochemical localization of the alpha 1A subunit of the P/Q-type calcium channel in the rat cerebellum

Among various types of low- and high-threshold calcium channels, the high voltage-activated P/Q-type channel is the most abundant in the cerebellum. These P/Q-type channels are involved in the regulation of neurotransmitter release and in the integration of dendritic inputs. We used an antibody specific for the alpha1A subunit of the P/Q-type channel in quantitative pre-embedding immunogold labelling combined with three-dimensional reconstruction to reveal the subcellular distribution of pre- and postsynaptic P/Q-type channels in the rat cerebellum. At the light microscopic level, immunoreactivity for the alpha1A protein was prevalent in the molecular layer, whereas immunostaining was moderate in the somata of Purkinje cells and weak in the granule cell layer. At the electron microscopic level, the most intense immunoreactivity for the alpha1A subunit was found in the presynaptic active zone of parallel fibre varicosities. The dendritic spines of Purkinje cells were also strongly labelled with the highest density of immunoparticles detected within 180 nm from the edge of the asymmetrical parallel fibre-Purkinje cell synapses. By contrast, the immunolabelling was sparse in climbing fibre varicosities and axon terminals of GABAergic cells, and weak and diffuse in dendritic shafts of Purkinje cells. The association of the alpha1A subunit with the glutamatergic parallel fibre-Purkinje cell synapses suggests that presynaptic channels have a major role in the mediation of excitatory neurotransmission, whereas postsynaptic channels are likely to be involved in depolarization-induced generation of local calcium transients in Purkinje cells.     

Effects of hypocretin2-saporin and antidopamine-beta-hydroxylase-saporin neurotoxic lesions of the dorsolateral pons on sleep and muscle tone

The hypocretin neurons have been implicated in regulating sleep-wake states as they are lost in patients with the sleep disorder narcolepsy. Hypocretin (HCRT) neurons are located only in the perifornical region of the posterior hypothalamus and heavily innervate pontine brainstem neurons, such as the locus coeruleus (LC), which have traditionally been implicated in promoting arousal. It is not known how the hypocretin innervation of the pons regulates sleep-wake states as pontine lesions have never been shown to increase sleep. It is likely that in previous studies specific neurons were not lesioned. Therefore, in this study, we applied saporin-based neurotoxins to the dorsolateral pons and monitored sleep in rats. Anti-dopamine-beta-hydroxylase-saporin killed the LC neurons but sleep was affected only during a two hour light-dark transition period. Application of hypocretin2-saporin killed fewer LC neurons relative to other adjacent neurons. This occurred because the LC neurons possess the hypocretin receptor 1 but the ligand hypocretin 2 binds to this receptor with less affinity relative to the hypocretin receptor 2. The hypocretin2-saporin lesioned rats compared to controls had increased sleep during the dark period and displayed increased limb movements during REM sleep. None of the lesioned rats had sleep onset REM sleep periods or cataplexy. We conclude that the hypocretin innervation to the pons functions to awaken the animal when the lights turn off (via its innervation of the LC), sustains arousal and represses sleep during the rest of the night (via a wider innervation of other pontine neurons), and modulates muscle tone.     

Experience-dependent changes in the hippocampus of domestic chicks: a model for spatial memory

In the domestic chicken, providing visual barriers for a brief period early in life has been found to improve spatial memory [R. Freire et al. (2004)Animal Behaviour, 67, 141-150]. In the present study we compared the structure of neurons in the hippocampus and neostriatum in chicks reared with or without visual barriers. From 8 to 16 days of age, chicks were reared in pens either with two wooden screens (Treatment E) or with no screens (Treatment C). At 16 days of age, chicks were anaesthetized, perfused intracardially and brain samples collected and stained using a Golgi-Cox technique. Morphometric analysis revealed that the multipolar projection neurons of Treatment E chicks had longer dendrites (ANOVA, F(1,14) = 7.4, P < 0.05) and had more spines per 20 micro m of dendrite (SLD; ANOVA, F(1,14) = 10.6, P < 0.01) than those of Treatment C chicks. By contrast, no evidence was found that rearing treatment differentially influences dendrite length or SLD in the neostriatum, suggesting that the above environment-induced changes may be specific to the hippocampus. Multipolar projection neuron dendrites of the right hemisphere were longer (ANOVA, F(1,14) = 36.4, P < 0.0001) and had more spines (ANOVA, F(1,14) = 8.8, P < 0.05) than dendrites of the left hemisphere, supporting previous findings that the right hemisphere of chickens is predominantly involved in spatial processing. We conclude that the chicken provides a useful model for the study of developmental plasticity in brain and behaviour, partly because the possibility of rearing chicks in isolation and imprinting them on an artificial object provides a means of accurately manipulating early experience.     

Adenosine A2A receptors are involved in physical dependence and place conditioning induced by THC

A2A adenosine and CB1 cannabinoid receptors are highly expressed in the central nervous system, where they modulate numerous physiological processes including adaptive responses to drugs of abuse. Both purinergic and cannabinoid systems interact with dopamine neurotransmission (through A2A and CB1 receptors, respectively). Changes in dopamine neurotransmission play an important role in addictive-related behaviours. In this study, we investigated the contribution of A2A adenosine receptors in several behavioural responses of Delta9-tetrahydrocannabinol (THC) related to its addictive properties, including tolerance, physical dependence and motivational effects. For this purpose, we first investigated acute THC responses in mice lacking A2A adenosine receptors. Antinociception, hypolocomotion and hypothermia induced by acute THC administration remained unaffected in mutant mice. Chronic THC treatment developed similar tolerance to these acute effects in wild-type and A2A-knockout mice. However, differences in the body weight pattern were found between genotypes during such chronic treatment. Interestingly, the somatic manifestations of SR141716A-precipitated THC withdrawal were significantly attenuated in mutant mice. The motivational responses of THC were also evaluated by using the place-conditioning paradigm. A significant reduction of THC-induced rewarding and aversive effects was found in mice lacking A2A adenosine receptors in comparison with wild-type littermates. Binding studies revealed that these behavioural changes were not associated with any modification in the distribution and/or functional activity of CB1 receptors in knockout mice. Therefore, this study shows, for the first time, a specific involvement of A2A receptors in the addictive-related properties of cannabinoids.     

Clinical pharmacokinetics of amfetamine and related substances: monitoring in conventional and non-conventional matrices

Consumption of amfetamine-type stimulants, including classical amfetamines and 'designer drugs', has been recognised as one of the most significant trends in drug abuse at the end of the past century and at the beginning of the current one. The first cause is the increasing consumption amongst youth of methylenedioxy- and methoxy-substituted amfetamines, of which the pharmacology in humans is currently under investigation. Secondly, the abuse of more classical amfetamines, such as amfetamine itself and metamfetamine, continues to be highly prevalent in some geographical regions. Amfetamines are powerful psychostimulants, producing increased alertness, wakefulness, insomnia, energy and self-confidence in association with decreased fatigue and appetite as well as enhanced mood, well-being and euphoria. From a clinical pharmacokinetic perspective, amfetamine-type stimulants are rather homogeneous. Their oral bioavailability is good, with a high distribution volume (4 L/kg) and low binding to plasma proteins (less than 20%). The elimination half-life is 6-12 hours. Both hepatic and renal clearance contribute to their elimination from the body. Hepatic metabolism is extensive in most cases, but a significant percentage of the drug always remains unaltered. Amfetamine and related compounds are weak bases, with a pKa around 9.9, and a relatively low molecular weight. These characteristics allow amfetamine-type stimulants to diffuse easily across cell membranes and lipid layers and to those tissues or biological substrates with a more acidic pH than blood, facilitating their detection in alternative matrices at relatively high concentrations. In most cases, the concentrations found are higher than expected from the Henderson-Hasselbach equation. Drug monitoring in non-conventional biological matrices (e.g. saliva, hair, nails, sweat) has recently gained much attention because of its possible applications in clinical and forensic toxicology. An individual's past history of medication, compliance or drug abuse can be obtained from testing of hair and nails, whereas data on current status of drug use can be provided by analysis of sweat and saliva. Because of the physicochemical properties of amfetamine-type stimulants, this group of drugs is one of the most suitable for drug testing in non-conventional matrices.