Tomás A. Reader

Autoradiographic study of α1‐ and α2‐noradrenergic and serotonin1A receptors in the spinal cord of normal and chronically transected cats

Serotoninergic and noradrenergic drugs have been shown to initiate and/or modulate locomotion in cats after spinal cord transection and in patients suffering from spinal cord injuries. To establish a firmer basis for locomotor pharmacotherapy, the distribution of α1‐ and α2‐noradrenergic and serotonin1A (5‐HT1A) receptors was examined in the spinal cord of control cats and of from animals with spinal cord transection at T13 some weeks or months previously. In control cats, the highest levels of α1‐noradrenergic receptors, labeled with [3H]prazosin, were found in laminae II, IX, and X. The α2‐noradrenergic receptors, labeled with [3H]idazoxan, were found mainly in laminae II, III, and X, with moderate densities in lamina IX. After spinal transection, both receptors did not change in segments above the lesion. At 15 and 30 days after spinal transection, binding significantly increased in laminae II, III, IV, and X for α2 and in laminae I, II, III, and IX for α1 receptors in lumbar segments. For longer survival times, binding densities returned to near control values. The 5‐HT1A receptors, labeled with [3H]8‐hydroxy‐dipropylaminotetralin, were found mainly in laminae I–IV and X. After spinal transection, binding significantly increased only in laminae II, III, and X of lumbar segments at 15 and 30 days. Thereafter, binding returned to control values. The pronounced upregulation of different monoaminergic receptors observed in the lumbar region in the first month after spinal transection suggests that these receptors may be important during the period when cats normally recover functions such as locomotion of the hindlimbs. J. Comp. Neurol. 406:402–414, 1999.

Serotonin 5-HT1 and 5-HT2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study

Neonatal destruction of nigrostriatal dopamine neurons by cerebroventricular injection of 6-hydroxydopamine (6-OHDA) results in a serotonin (5-HT) hyperinnervation of the rostral neostriatum in adult rat. Quantitative ligand-binding autoradiography was used to compare the density of various 5-HT receptor subtypes in the adult brain of control and neonatally 6-OHDA-lesioned rats. 5-HT1A, 5-HT1B, 5HT1nonAB and 5-HT2 sites were labeled with [3H]8-OH-DPAT, [125I]cyanopindolol, [3H]5-HT and [125I]DOI, respectively, and measured in the rostral and caudal halves of neostriatum and selected forebrain or midbrain regions. 5-HT1A binding, measured after 6 months, was unchanged in all regions examined including the dorsal raphe nucleus. Three months after the lesion, 5-HT1B binding was increased throughout the neostriatum (30%), but also in the substantia nigra (50%) and globus pallidus (30%), suggesting an up-regulation and an increased axonal transport of these receptors in neostriatal projection neurons. 5-HT1nonAB binding was also increased throughout the neostriatum (40%) and in the substantia nigra (50%), but unchanged in the globus pallidus, as if this up-regulation preferentially involved striatonigral as opposed to striatopallidal neurons. 5-HT2 binding showed an even greater increase (60%), which was restricted to the rostral half of neostriatum and also seemed imputable to an up-regulation as heteroreceptors. Even though the exact cause(s) of these receptor increases could not be determined, their anatomical distribution suggested that they were somehow related to the initial dopamine denervation in the case of the 5-HT1B and 5-HT1nonAB receptors, and more tightly linked to the 5-HT hyperinnervation in the case of the 5-HT2 receptors. Such receptor changes could participate in adaptive mechanisms implicating other transmitters and behavioral disturbances observed in this particular experimental model. Interestingly, they could also account for an enhancement of neostriatal 5-HT function even in a condition where extracellular levels of 5-HT apparently remain normal because of increased uptake.

Catecholamines released from cerebral cortex in the cat; decrease during sensory stimulation.

In an attempt to determine the functional role of catecholamine (CA) nerve terminals in cerebral cortex the release of endogenous norepinephrine (NE) and dopamine (DA) into superfusates from visual and somatosensory cortex of the cat have been measured by a sensitive radiometric enzymatic assay based on the methylation of CA by catechol-O-methyltransferase (COMT) in the presence of a [3H]-methyl donor and followed by resolution of 3H derivatives through a series of organic extractions. In the flaxedilized animal maintained under local anaesthesia with artificial respiration the concentration of CA measured in 30-min superfusates was fairly constant in a given experiment under basal conditions without sensory stimulation, but varied widely from one experiment to another. Variations in NE were often independent of those for DA. For visual cortex the average basal release of NE in experiments was 20.09 +/- 3.64 pg/min/sq.cm while the average for DA was 34.01 +/- 7.62 pg/min/sq.cm. In all experiments intermittent visual stimulation (15/sec) produced a significant reduction in release rate averaging about 42% for NE and 64% for DA in visual cortex. The reduction was relatively non-specific since visual or somatic sensory stimulation produced a decrease in release from both visual and somatic sensory cortical areas. Since it has been shown that there is a relatively non-specific increase in acetylcholine (ACh) release from sensory cortex during stimulation, it is proposed that ACh may regulate CA release at presynaptic CA terminals in the cortex as it does in the periphery. A marked increase in CA release observed on perfusing with nicotine or atropine is consistent with this hypothesis.

Pharmacological aids to locomotor training after spinal injury in the cat

This Topical Review summarizes some of the work we have done mainly in the cat using agonists and antagonists of various neurotransmitter systems injected intravenously or intrathecally to initiate or modulate the expression of hindlimb locomotion after a spinal lesion at T13. The effects of the same drugs are compared in various preparations: complete spinal, partial spinal or intact cats. This has revealed that there can be major differences in these effects. In turn, this suggests that although the locomotor rhythm might normally be triggered and modulated by the activation of a variety of receptors (noradrenaline, serotonin, glutamate), after spinalization there appears to be a predominance of glutamatergic mechanisms. Recent work also suggests that, in the cat, the integrity of the midlumbar segments is crucial for the expression of spinal locomotion. Taken together, this work raises some hope that a targeted pharmacotherapy with better understood drugs and mode and locus of delivery could become a clinical reality.

Pharmacological Activation and Modulation of the Central Pattern Generator for Locomotion in the Cata

Abstract: Pharmacological agents have been shown to be capable of inducing a pattern of rhythmic activity recorded in muscle nerves or motoneurons of paralyzed spinal cats that closely resembles the locomotor pattern seen in intact cats. Further work, using intraperitoneal or intrathecal injections, suggests that different neurotransmitters may be involved in various aspects of locomotor control, e.g., initiation and modulation of the pattern. Although precursors, agonists or the neurotransmitters themselves of several systems have been investigated (noradrenergic, dopaminergic, serotonergic, glutamatergic), the noradrenergic system seems the most efficient in triggering locomotion in complete spinal cats, with the α‐2 agonists (clonidine, tizanidine, oxymetazoline) being more potent than the α‐1 agonist, methoxamine. Moreover, the potency of the drugs may depend on the time of application after the spinal lesion. In chronic spinal cats capable of spontaneous walking on hindlimbs on the treadmill, all neurotransmitters appear to exert distinct recognizable effects on the locomotor pattern. More recent work also suggests that the effects of drugs may differ significantly depending on the type of spinal lesion. For instance, clonidine further reduces the level of weight support during quadrupedal locomotion of cats with lesions of the ventral‐ventrolateral funiculi, possibly due to an interference of clonidine with essential compensatory mechanisms used by these animals to walk. Such considerations as the type of drugs, type of lesions, and the time after the lesion will be important for future studies in spinal cord injured patients.

Monoamines and metabolites in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

Alterations in the metabolism of monoamine neurotransmitters have been proposed to be involved in the development of the hepatic encephalopathy (HE) associated with experimental and human liver failure. In order to evaluate this hypothesis, the monoamines and some of their metabolites were measured in homogenates of caudate nucleus (CAU), prefrontal (PFCo) and frontal cortex (FCo) dissected from brains obtained at autopsy from nine cirrhotic patients who had died in hepatic coma and an equal number of control subjects, free from neurological, psychiatric and hepatic disorders, matched for age and time interval from death to freezing of autopsied brain samples. Monoamine measurements were performed by high-performance liquid chromatography with ion-pairing and electrochemical detection after a simple extraction procedure. In all three regions investigated, concentrations of dopamine (DA) were unchanged in cirrhotic patients vs controls while its metabolites, 3-methoxytyramine (3-MT) and homovanillic acid (HVA) were selectively affected i.e.3-MT was found to be increased in CAU, while HVA levels were increased in FCo and CAU. DOPAC was also found to be unchanged in CAU. Noradrenaline (NA) levels were greatly increased in PFCo and FCo of cirrhotic patients but remained unchanged in CAU. No significant differences in the concentrations of either serotonin (5-HT) or of its precursor 5-hydroxytryptophan (5-HTP) were found in any of the three regions studied. However, 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, was increased in PFCo and CAU of cirrhotic patients. These findings show that selective alterations of catecholamine and 5-HT systems are involved in human HE and therefore, they may play an important role in the pathogenesis of certain neurological symptoms associated with this encephalopathy.

Altered Serotonin and Dopamine Metabolism in the CNS of Serotonin 5‐HT1A or 5‐HT1B Receptor Knockout Mice

Abstract: Measurements of serotonin (5‐HT), dopamine (DA), andnoradrenaline, and of 5‐HT and DA metabolites, were obtained by HPLC from 16brain regions and the spinal cord of 5‐HT1A or 5‐HT1Bknockout and wild‐type mice of the 129/Sv strain. In 5‐HT1Aknockouts, 5‐HT concentrations were unchanged throughout, but levels of 5‐HTmetabolites were higher than those of the wild type in dorsal/medial raphenuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was takenas an indication of increased 5‐HT turnover, reflecting an augmented basalactivity of midbrain raphe neurons and consequent increase in theirsomatodendritic and axon terminal release of 5‐HT. It provided a likelyexplanation for the increased anxious‐like behavior observed in5‐HT1A knockout mice. Concomitant increases in DA content and/or DAturnover were interpreted as the result of a disinhibition of DA, whereasincreases in noradrenaline concentration in some territories of projection ofthe locus coeruleus could reflect a diminished activity of its neurons. In5‐HT1B knockouts, 5‐HT concentrations were lower than those of thewild type in nucleus accumbens, locus coeruleus, spinal cord, and probablyalso several other territories of 5‐HT innervation. A decrease in DA,associated with increased DA turnover, was measured in nucleus accumbens.These changes in 5‐HT and DA metabolism were consistent with the increasedaggressiveness and the supersensitivity to cocaine reported in5‐HT1B knockout mice. Thus, markedly different alterations in CNSmonoamine metabolism may contribute to the opposite behavioral phenotypes ofthese two knockouts.

[3H]paroxetine binding and serotonin content of rat cortical areas, hippocampus, neostriatum, ventral mesencephalic tegmentum, and midbrain raphe nuclei region following p-chlorophenylalanine and p-chloroamphetamine treatment.

Abstract: The agents p‐chlorophenylalanine (PCPA) and p‐chloroamphetamine (PCA) deplete brain serotonin (5‐HT) levels by two different mechanisms; PCPA inhibits the enzyme tryptophan hydroxylase, whereas PCA has a neurotoxic action on certain 5‐HT neurons. The parameters of [3H]paroxetine binding to homogenates prepared from the cerebral cortex of rats treated with PCPA, PCA, or saline vehicle were investigated. The tissue concentrations of 5‐HT and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were also determined by HPLC in the same brain samples. After PCPA treatment, neither the maximum binding capacity (Bmax) nor the dissociation constant (KD) of [3H]paroxetine for the 5‐HT uptake recognition site differed from controls despite a substantial reduction in the concentration of 5‐HT and 5‐HIAA. In contrast, significant changes in both the Bmax and KD values were observed in the cerebral cortex of rats treated with PCA. Furthermore, [3H]paroxetine binding and tissue concentrations of 5‐HT and 5‐HIAA were measured in the following different regions of the rat brain: cingulate, parietal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum; ventral mesencephalic tegmentum; and midbrain raphe nuclei region after administration of PCPA, PCA, or saline vehicle. There was an excellent correlation between regional 5‐HT levels and specific [3H]paroxetine binding in control and PCA‐treated rats although this correlation was lost after PCPA treatment. Under these conditions, the 5‐HT innervation remains unchanged whereas the concentration of 5‐HT and 5‐HIAA is greatly reduced. Thus, [3H]paroxetine binding appears to provide a reliable marker of 5‐HT innervation density within the mammalian CNS.

ELEVATION OF DOPAMINE D2 BUT NOT D1 RECEPTORS IN ADULT RAT NEOSTRIATUM AFTER NEONATAL 6-HYDROXYDOPAMINE DENERVATION

Monoamine levels and the binding properties of [3H]SCH23390, a D1-specific ligand, and [3H]raclopride, a D2-specific ligand, were measured in the rostal and caudal neostriatum to investigate the fate of dopamine receptors following bilateral cerebroventricular injection of 6-hydroxydopamine in 3-day-old rats. After survival times of 15, 30 or 90 days, measurement of monoamine levels and of [3H]SCH23390 binding were also obtained from the cerebral cortex. At all three survival times, dopamine content was reduced by more than 90% of control values in both the rostral and caudal neostriatum; in cerebral cortex, the dopamine depletion was less profound (80%) and noticeable only after 1 and 3 months. In the rostral but not the caudal neostriatum, serotonin and 5-hydroxyindoleacetic acid concentrations were markedly increased at 1 and 3 months; cortical serotonin also was augmented at 3 months. There were no changes in neostriatal [3H]SCH23390 binding at any of the survival times, but a transient elevation occurred in the cortex at 1 month. In the rostral but not the caudal neostriatum, [3H]raclopride binding showed a slight elevation at 1 month and a further, highly significant increase at 3 months. As measured in individual rats, this increase in [3H]raclopride binding was linearly correlated with the increase in serotonin turnover (ratio of 5-hydroxyindoleacetic acid/serotonin). Such an up-regulation of D2 receptors, restricted to the rostral neostriatum which was also the site of a serotonin hyperinnervation, was probably indicative of a serotonin control on the expression of D2 receptors after dopamine denervation.

Serotonin Neural Adaptations to Ontogenetic Loss of Dopamine Neurons in Rat Brain

Abstract: In rat, the neonatal destruction of nigrostriatal dopamine (DA) neurons by intracerebral administration of 6‐hydroxydopamine entails dramatic changes in serotonin (5‐hydroxytryptamine, 5‐HT) as well as DA function. Most striking is the 5‐HT hyperinnervation of the adult neostriatum, associated with increases in density of various 5‐HT receptor subtypes and enhanced neuronal responsiveness to the iontophoretic application of 5‐HT and its 5‐HT1B/2C and 5‐HT2A/2C receptor agonists, m‐chlorophenylpiperazine and iododimethoxyphenylaminopropane. The topographical distribution of these changes is consistent with up‐regulation and/or increased production and transport of 5‐HT1B and 5‐HT2A receptors by the neostriatal projection neurons, as confirmed for the 5‐HT2A receptor in a recent in situ hybridization study. It is interesting that this study has also shown that increases in both 5‐HT2A binding and mRNA level were abolished by chronic pretreatment with the DA agonists, apomorphine and SKF 38393, suggesting a regulatory influence of DA in the expression of this 5‐HT receptor. D1 receptor binding is known to be slightly reduced in the rostral neostriatum of these rats, a down‐regulation apparently imputable to a reduced rate of synthesis of the receptor. In contrast, D2 receptor binding is increased throughout the DA‐denervated and 5‐HT‐hyperinnervated neostriatum, perhaps due to some post‐transcriptional modifications. Stereotyped and motor behaviors induced by systemic treatment with D1 and D2 agonists are markedly enhanced in these rats (behavioral supersensitivity), although priming is commonly required to unmask a latent D1 supersensitivity. In the case of oral activity, however, overt behavioral supersensitivity is induced by D1 as well as D2 agonists. Moreover, there is overt supersensitivity of oral activity in response to the 5‐HT receptor agonist m‐chlorophenylpiperazine, which is presumably imputable to 5‐HT2C receptors and may be demonstrated even in the absence of supersensitivity to D1 receptor agonist. 5‐HT adaptations, therefore, seem to play a role not only in the abnormal spontaneous behavior, but also in the behavioral supersensitivity to 5‐HT as well as DA receptor agonists in these rats.