No abstract

1 The present study examined 5-HT 2C receptor agonist-induced behavioural tolerance and 5-HT 2C receptor down-regulation in adult rat brain. The e ect of chronic subcutaneous infusion of the 5-HT 2C receptor agonist, m-chlorophenylpiperazine (m-CPP, 10 mg kg 71 , day 71 ), for 14 days was examined on daily food intake, the ability of acute m-CPP (2.5 mg kg 71 , i.p.) to induce hypolocomotion in a novel arena and elevate plasma corticosterone levels and on ex vivo cortical [ 3 H]-mesulergine binding and hippocampal 5-HT 2C receptor protein levels. 2 Before chronic infusion, m-CPP (2.5 mg kg 71 , i.p.) attenuated the number of turns and rears made in a novel open ®eld arena. In contrast, while m-CPP still elicited this hypolocomotion following 14 days, saline infusion, no such hypolocomotion occurred in rats given chronic m-CPP (10 mg kg 71 day 71 ), indicating that almost complete tachyphylaxis of this behaviour occurred with chronic 5-HT 2C receptor agonist injection. 3 During chronic infusion of m-CPP, rats consumed less food per day than saline-treated controls. Acute challenge with m-CPP following two weeks, treatment still attenuated food intake over the next four hours (by 43% and 30%, respectively from that on the previous day) in saline and m-CPP infusion groups, showing that only partial tolerance to 5-HT 2C receptor agonist-induced hypophagia occurred. 4 In naive home cage rats, plasma corticosterone was elevated in a dose-dependent manner 35 min after m-CPP injection (0.5, 1 and 3 mg kg 71 , i.p.) but levels were comparable to control values 16 h after m-CPP (2, 5 and 10 mg kg 71 , i.p.). Sixteen hours after a single m-CPP injection (2.5 mg kg 71 , i.p.), plasma corticosterone levels were comparable in a group of rats which had received 14 days infusion of m-CPP or saline. However, following a similar acute m-CPP injection (2.5 mg kg 71 , i.p., 716 h) in rats previously infused for 14 days with m-CPP, plasma corticosterone levels were lower than those in a separate group which received no chronic infusions (but only acute m-CPP injection), even though the plasma m-CPP levels were comparable in both groups. The data are consistent with the proposal that chronic m-CPP induced some down-regulation of hypothalamic 5-HT 2C receptors which contribute, in a tonic manner, to plasma corticosterone secretion under the conditions investigated. 5 Chronic m-CPP infusion reduced the amount of [ 3 H]-mesulergine binding (by 27%, without altering the K D ) in membranes prepared from parietal/occipital/temporal cortex (under conditions to exclude binding to 5-HT 2A receptors) and 5-HT 2C receptor protein-like immunoreactive levels measured by radioimmunoassay in the hippocampus by 38%, con®rming that 5-HT 2C receptor down-regulation had occurred. 6 Even after 14 days m-CPP infusion only partial behavioural tolerance and 5-HT 2C receptor downregulation were observed, which may vary in di erent brain regions of the rat. Thus the hypophagia produced by m-CPP may involve activation of 5-HT 2C receptors in the hypothalamus,...

A synthetic peptide, corresponding to the N-terminal decapeptide (+Y11C12) of the rat 5-hydroxytryptamine2C (5-HT2C) receptor protein was used to produce a sheep polyclonal antiserum. Western blot analysis showed that the resultant antibody G241 recognised two membrane proteins, one (55 kDa) approximating the molecular mass of the 5-HT2C receptor (52 kDa) and a second (63 kDa), which may be a glycosylated form of the receptor protein. HEK 293 cells transfected with human 5-HT2C cDNA displayed intense cell surface immunoreactivity with the 5-HT2C antiserum, which was completely prevented by incubating the antibody with the synthetic 5-HT2C peptide (10 microM), whilst neither non-immune serum nor untransfected cells displayed any immunoreactivity. A radioimmunoassay was developed to quantify the regional distribution of 5-HT2C-like immunoreactivity (LI) in the adult rat brain. The choroid plexus contained five-fold higher levels of 5-HT2C-LI than any brain region but high levels were found in the frontal cortex, septum, hypothalamus, and striatum, intermediate levels in the thalamus and midbrain, and lower levels in brainstem, cerebellum, and spinal cord. In rat cortical membranes, the B(max) value from [3H]-mesulergine binding was ten-fold lower than 5-HT2C-LI levels determined by radioimmunoassay, which may reflect measurement of internalised receptor protein by radioimmunoassay which is not detected with conventional 5-HT2C ligands. Ten days after depletion of 5-HT with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), there was a significant increase in 5-HT2C-LI in the choroid plexus and the ventral cervical spinal cord, suggesting that receptors therein are located post-synaptic to destroyed serotonergic nerve terminals. In contrast, the significant reduction in 5-HT2C-LI observed in the midbrain, brainstem, and dorsal thoracic spinal cord following 5,7-DHT implies that 5-HT2C receptors may be located on 5-HT nerve terminals in these regions.

A synthetic peptide, corresponding to the N-terminal decapeptide (+Y11C12) of the rat 5-hydroxytryptamine2C (5-HT2C) receptor protein was used to produce a sheep polyclonal antiserum. Western blot analysis showed that the resultant antibody G241 recognised two membrane proteins, one (55 kDa) approximating the molecular mass of the 5-HT2C receptor (52 kDa) and a second (63 kDa), which may be a glycosylated form of the receptor protein. HEK 293 cells transfected with human 5-HT2C cDNA displayed intense cell surface immunoreactivity with the 5-HT2C antiserum, which was completely prevented by incubating the antibody with the synthetic 5-HT2C peptide (10 microM), whilst neither non-immune serum nor untransfected cells displayed any immunoreactivity. A radioimmunoassay was developed to quantify the regional distribution of 5-HT2C-like immunoreactivity (LI) in the adult rat brain. The choroid plexus contained five-fold higher levels of 5-HT2C-LI than any brain region but high levels were found in the frontal cortex, septum, hypothalamus, and striatum, intermediate levels in the thalamus and midbrain, and lower levels in brainstem, cerebellum, and spinal cord. In rat cortical membranes, the B(max) value from [3H]-mesulergine binding was ten-fold lower than 5-HT2C-LI levels determined by radioimmunoassay, which may reflect measurement of internalised receptor protein by radioimmunoassay which is not detected with conventional 5-HT2C ligands. Ten days after depletion of 5-HT with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), there was a significant increase in 5-HT2C-LI in the choroid plexus and the ventral cervical spinal cord, suggesting that receptors therein are located post-synaptic to destroyed serotonergic nerve terminals. In contrast, the significant reduction in 5-HT2C-LI observed in the midbrain, brainstem, and dorsal thoracic spinal cord following 5,7-DHT implies that 5-HT2C receptors may be located on 5-HT nerve terminals in these regions.