G.W. Bennett

The effects of monoamine neurotoxins on peptides in the rat spinal cord

The coexistence of two neuronally-localised peptides, substance P and thyrotropin-releasing hormone (TRH), in descending serotoninergic nerve fibres to the spinal cord was investigated using immunocytochemical and biochemical methods. Substance P-like material in the spinal cord was shown to be identical to the undecapeptide substance P by the criteria of gel filtration, high performance liquid chromatography and behaviour in substance P specific radioimmunoassays. Immunocytochemical staining for 5-hydroxytryptamine, substance P, and TRH showed that all three substances had a similar distribution in nerve fibres and terminals in the ventral and lateral grey matter of the spinal cord. After treatment with the serotonin neurotoxin 5,7-dihydroxytryptamine, neuronal elements containing 5-hydroxytryptamine, substance P and TRH degenerated and disappeared from these parts of the spinal cord in parallel with one another. Biochemical measurements of 5-hydroxytryptamine, substance P and TRH in the spinal cord after treatment with 5,7-dihydroxytryptamine confirmed that these three substances were all depleted from the ventral horn and, in addition, showed that there was a small depletion of substance P from the dorsal horn. Two other neuropeptides, somatostatin and methionine-enkephalin were not depleted from the spinal cord by treatment with 5,7-dihydroxytryptamine nor was substance P in other parts of the brain. Substance P in the spinal cord was unaffected by 6-hydroxydopamine, a drug known to destroy catecholamine-containing neurones. These results are consistent with coexistence of substance P and TRH together with 5-hydroxytryptamine in the descending axons and terminals of bulbospinal neurones.

5‐Fluorouracil chemotherapy affects spatial working memory and newborn neurons in the adult rat hippocampus

Chemotherapy‐associated memory deficits in adults are prevalent with systemic treatment utilizing 5‐fluorouracil (5‐Fu). 5‐Fu disrupts cell proliferation and readily crosses the blood–brain barrier. Proliferating cells within the adult dentate gyrus of the hippocampus give rise to new neurons involved in memory and learning and require neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) to nurture this process of adult neurogenesis. Some of these proliferating cells are anatomically and functionally supported by vascular endothelial cells. We propose that systemically administered 5‐Fu chemotherapy will cause deficits in hippocampal memory that are associated with altered BDNF levels and proliferating cells (particularly vascular‐associated cells) in the dentate gyrus. This was tested by determining the effect of 5‐Fu on spatial working memory as modelled by the object location recognition test. Numbers of vascular‐associated (VA) and non‐vascular‐associated (NVA) proliferating cells in the dentate gyrus were measured using double‐labelling immunohistochemistry with markers of proliferation (Ki67) and endothelial cells (RECA‐1). 5‐Fu‐induced changes in hippocampal BDNF and doublecortin (DCX) protein levels were quantified using Western immunoblotting. 5‐Fu chemotherapy caused a marginal disruption in spatial working memory and did not alter the total proliferating cell counts or the percentage of VA and NVA proliferating cells in the dentate gyrus. In contrast, 5‐Fu significantly reduced BDNF and DCX levels in the hippocampus, indicating alterations in neurotrophin levels and neurogenesis. These findings highlight the usefulness of animal models of ‘chemobrain’ for understanding the mechanisms that underlie chemotherapy‐associated declines in cognitive performance and memory.

Changes in monoamine metabolites measured by simultaneousin vivo differential pulse voltammetry and intracerebral dialysis

A direct comparison has been made of the drug-induced changes in extracellular levels of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid measured using intracerebral dialysis and differential pulse voltammetry with carbon fibre electrodes. The comparison was carried out in chloral hydrate anaesthetized rats with a pretreated carbon fibre electrode implanted in one striatum and an intracerebral dialysis loop in the contralateral striatum. 3,4-Dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in the dialysis perfusates were assayed by high pressure liquid chromatography with electrochemical detection. d-Amphetamine (2 mg/kg) decreased extracellular 3,4-dihydroxyphenylacetic acid and the height of the 3,4-dihydroxyphenylacetic acid oxidation peak, while haloperidol (0.5 mg/kg) increased 3,4-dihydroxyphenylacetic acid levels measured in the perfusates and the height of the 3,4-dihydroxyphenylacetic acid oxidation peak. In these experiments there were parallel changes in 3,4-dihydroxyphenylacetic acid levels and peak height and a close correlation between these changes. Tranylcypromine (10 mg/kg) produced an almost parallel decrease in extracellular 5-hydroxyindoleacetic acid (dialysis) and the height of the 5-hydroxyindoleacetic acid oxidation peak, with similar percentage changes and good correlation values being obtained. However, while 5-hydroxy-L-tryptophan (25 mg/kg) increased both the 5-hydroxyindoleacetic acid levels and the height of the 5-hydroxyindoleacetic acid oxidation peak, 5-hydroxyindoleacetic acid in the dialysis perfusates showed a greater increase than the oxidation peak. The results show a close correlation between changes in extracellular 3,4-dihydroxyphenylacetic acid and its respective voltammetric peak and strongly support the use of in vivo differential pulse voltammetry for monitoring dopamine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotrophin-releasing hormone analogues increase dopamine release from slices of rat brain.

Abstract: Rat brain slices were incubated with a high concentration of K+, thyrotrophin‐releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K+ (30 mM) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (105–10−3M) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose‐dependent fashion, whereas RX 77368 (10−4M) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.

Valproic acid reduces spatial working memory and cell proliferation in the hippocampus

Valproic acid (VPA) is widely used clinically, as an anticonvulsant and mood stabilizer but is, however, also known to block cell proliferation through its ability to inhibit histone deacetylase enzymes. There have been a number of reports of cognitive impairments in patients taking VPA. In this investigation we examined the relationship between cognition and changes in cell proliferation within the hippocampus, a brain region where continued formation of new neurons is associated with learning and memory. Treatment of rats by i.p. injection of VPA, reduced cell proliferation in the sub granular zone of the dentate gyrus within the hippocampus. This was linked to a significant impairment in their ability to perform a hippocampus-dependent spatial memory test (novel object location). In addition, drug treatment caused a significant reduction in brain-derived neurotrophic factor (BDNF) and Notch 1 but not doublecortin levels within the hippocampus. These results support the idea that VPA may cause cognitive impairment and provide a possible mechanism for this by reducing neurogenesis within the hippocampus.

Fluoxetine improves the memory deficits caused by the chemotherapy agent 5-fluorouracil

Cancer patients who have been treated with systemic adjuvant chemotherapy have described experiencing deteriorations in cognition. A widely used chemotherapeutic agent, 5-fluorouracil (5-FU), readily crosses the blood-brain barrier and so could have a direct effect on brain function. In particular this anti mitotic drug could reduce cell proliferation in the neurogenic regions of the adult brain. In contrast reports indicate that hippocampal dependent neurogenesis and cognition are enhanced by the SSRI antidepressant Fluoxetine. In this investigation the behavioural effects of chronic (two week) treatment with 5-FU and (three weeks) with Fluoxetine either separately or in combination with 5-FU were tested on adult Lister hooded rats. Behavioural effects were tested using a context dependent conditioned emotional response test (CER) which showed that animals treated with 5-FU had a significant reduction in freezing time compared to controls. A separate group of animals was tested using a hippocampal dependent spatial working memory test, the object location recognition test (OLR). Animals treated only with 5-FU showed significant deficits in their ability to carry out the OLR task but co administration of Fluoxetine improved their performance. 5-FU chemotherapy caused a significant reduction in the number of proliferating cells in the sub granular zone of the dentate gyrus compared to controls. This reduction was eliminated when Fluoxetine was co administered with 5-FU. Fluoxetine on its own had no effect on proliferating cell number or behaviour. These findings suggest that 5-FU can negatively affect both cell proliferation and hippocampal dependent working memory and that these deficits can be reversed by the simultaneous administration of the antidepressant Fluoxetine.

EFFECT OF NEUROPEPTIDES ON COGNITIVE FUNCTION

Recent evidence indicates that, in addition to the involvement of cholinergic and other neurotransmitter systems, various neuropeptides that occur in cortical and subcortical brain regions have a role in cognitive behavior. This evidence results largely from behavioral studies in rodents and other animals, following peptide administration and only in a very few cases from similar studies in human subjects. Several neuropeptides studied appear to enhance or produce changes conducive to improvement in cognitive performance and these include vasopressin, corticotrophin-releasing hormone (CRH), somatostatin, substance P, neuropeptide Y, and thyrotrophin-releasing hormone (TRH), while one peptide, galanin, has been reported to inhibit cognitive processes. Of those neuropeptides that improve performance, only TRH has been shown recently to attenuate the memory impairment of human subjects and Alzheimer patients treated with an anticholinergic drug, and this review describes a series of complimentary studies in adult and aged rodents that contribute to our understanding of the possible mechanisms involved in the role of TRH in cognition.

Involvement of 5‐HT2 receptors in the behaviours produced by intrathecal administration of selected 5‐HT agonists and the TRH analogue (CG 3509) to rats

1 The behavioural effects of the intrathecal injection of a thyrotrophin‐releasing hormone (TRH) analogue l‐orotyl‐l‐histidyl‐prolineamide (CG 3509, 0.5 μg), the non‐selective 5‐HT1 and 5‐HT2 receptor agonist 5‐methoxy‐N,N′‐dimethyltryptamine (5‐MeODMT, 2–100 μg) and the selective 5‐HT2 receptor agonist 2,5‐dimethoxy‐α,4‐dimethyl‐benzene ethamine hydrochloride (DOM, 2–25 μg) were compared with the response of systemically administered 5‐MeODMT (2 mg kg−1, i.p.) in rats, to establish whether the agonist‐induced behaviours were mediated by bulbospinal 5‐HT1 or 5‐HT2 receptors. 2 Intrathecal injection of 5‐MeODMT or DOM produced dose‐related back muscle contractions (a previously undocumented behaviour) and wet‐dog shakes which were both markedly attenuated by ritanserin pretreatment (1 mg kg−1, i.p.) indicating the involvement of 5‐HT2 receptors. In contrast, reciprocal forepaw treading, flat body posture and Straub‐tail were evoked by 5‐MeODMT but not by DOM indicating that these behaviours were not produced by 5‐HT2 receptor activation alone. However, as ritanserin pretreatment reduced the reciprocal forepaw treading induced by intrathecal 5‐MeODMT, this behaviour may be facilitated by 5‐HT2 receptor activation. 3 Intrathecal 5,7‐dihydroxytryptamine (5,7‐DHT, 2 × 150 μg) treatment decreased thoraco‐lumbar spinal cord 5‐HT (‐95%) and potentiated the back muscle contractions produced by intrathecal DOM injection without altering the wet‐dog shake behaviour. None of the components of the 5‐HT syndrome produced by 5‐MeODMT (2 mg kg−1, i.p.), with the exception of a small increase in wet‐dog shakes, was significantly altered by intrathecal 5,7‐DHT (which reduced thoraco‐lumbar spinal cord 5‐HT by 84%). Taken together these data suggest that the only 5‐HT agonist‐induced behaviour mediated by the activation of 5‐HT2 receptors located postsynaptic to bulbospinal 5‐hydroxytryptaminergic (5‐HTergic) neurones was back muscle contractions. 4 The wet‐dog shake and forepaw licking behaviors produced by intrathecal CG 3509 (0.5 μg) were attenuated when ritanserin was administered intrathecally 30 min before, but not when it was given at the same time as CG 3509 and neither behaviour was altered by intrathecal 5,7‐DHT. This suggests that bulbospinal 5‐HTergic neurones are not involved in the production of these TRH analogue‐induced behaviours and that the 5‐HT2 receptors which mediate these behaviours are not located in the spinal cord.

Effects of (R)-α-methylhistamine and scopolamine on spatial learning in the rat assessed using a water maze

The effects of (R)-α-methylhistamine ((R)-α-MeHA, a selective H3-receptor agonist) and scopolamine (SCOP, a muscarinic antagonist) were investigated on spatial learning and memory in the rat (Hooded Lister) using a water maze (WM). (R)-α-MeHA treatment (6.3 and 10 mg/kg IP) had no apparent effect on spatial learning but did result in enhanced spatial recall at the higher dose, assessed by a transfer (probe) test after training. In contrast, SCOP (0.5 mg/kg IP) induced a learning and memory deficit measured both during and after training. In animals treated with (R)-α-MeHA and SCOP, (R)-α-MeHA partially (6.3 mg/kg) and completely (10 mg/kg) reversed the SCOP-induced deficit during the training phase, while in the post-training transfer test, (R)-α-MeHA (10 mg/kg) significantly reduced the SCOP-induced memory deficit. None of the treatments described resulted in impaired visual acuity as demonstrated by a raised platform test. These results are consistent with a role for histamine in cognitive processes and suggest a possible interaction between central histamine and cholinergic mechanisms associated with rodent spatial learning and memory.

The effects of 5,7-dihydroxytryptamine and p-chlorophenylalanine on thyrotrophin-releasing hormone in regions of the brain and spinal cord of the rat

The distribution of thyrotrophin-releasing hormone (TRH) and 5-hydroxytryptamine (5-HT) were compared in ten regions of the rat brain and in lumbar spinal cord. After dissection, using a cutting box and tissue punches, TRH was measured by radioimmunoassay and 5-HT by HPLC with electrochemical detection. Within the brain the highest levels of TRH were found in the median eminence and the remaining hypothalamus. There were also relatively high levels in the suprachiasmatic nucleus, septal nuclei and nucleus accumbens. Highest levels of 5-HT were found in the raphe nuclei, hypothalamic nuclei, nucleus accumbens, stria terminalis, septal nuclei and hippocampus. 5,7-Dihydroxytryptamine (5,7-DHT; 200 micrograms, i.c.v.) markedly reduced levels of 5-HT in brain and spinal cord. In the ventral lumbar cord there was a comparable decrease of TRH and 5-HT (-80%) and a smaller but significant decrease in the nucleus accumbens (-55%) and septal nuclei (-38%). p-Chlorophenylalanine (PCPA; 250 mg/kg X 2) reduced levels of 5-HT (-80%), without significantly altering those of noradrenaline. p-Chlorophenylalanine also significantly reduced levels of TRH in the nucleus accumbens (-72%) but not in the other regions of brain or spinal cord taken. The results are discussed in relation to the previously described co-existence of TRH and 5-HT in the spinal cord and the possible alternative forms of interactions between amine and peptide in the nucleus accumbens.