5-HT precursor loading, but not 5-HT receptor agonists, increases motor function after spinal cord contusion in adult rats.

Serotonergic (5-HT) receptors are upregulated following spinal cord transection. Stimulation by administration of serotonergic receptor agonists has been successful in improving hindlimb function. We tested whether this strategy would be successful in incomplete injury models (moderate or severe thoracic contusion) where descending projections are partially spared which should produce less denervation-induced receptor upregulation. Adult rats received midthoracic moderate (MOD: 25 mm drop) or severe (SEV: 50 mm drop) contusion injuries. Distribution of 5-HT and its transporter and expression of 5-HT(2C) receptors were evaluated in lumbar spinal cord and motor response to 5-HT receptor activation was assessed using open field locomotion (BBB) score, percent weight supported treadmill stepping (%WS) and evaluation of hindlimb muscle activation (tremor and serotonin syndrome). 5-HT immunostaining 3 months post-contusion revealed few 5-HT fibers caudal to the severe contusion, and more spared caudal to the moderate contusion. The distribution of 5-HT transporter paralleled 5-HT staining, but was more greatly reduced. Thus serotonin reuptake may be less efficient in the injured spinal cord. Immunostaining for the 5-HT(2C) receptor in the dorsal and ventral horns at L5 showed significant upregulation in SEV, compared to sham or MOD rats. Neither 5-HT(2C) nor 5-HT(1A) receptor agonists, alone or in combination, nor the serotonin transporter inhibitor d-fenfluramine modified BBB scores or %WS in either group. Despite the increased sensitivity of post-synaptic targets, agonist treatment did not improve function in SEV rats. We conclude that selective 5-HT(2C) or 5-HT(1A) receptor activation was not effective in improving hindlimb function after incomplete lesions. In contrast, the 5-HT precursor 5-hydroxytryptophan (L-5-HTP), which leads to activation of all classes of 5-HT receptors, increased both %WS and hindlimb activity in the MOD group. While no side effects were observed in normal or MOD rats, SEV rats displayed hindlimb tremors and 33% mortality, indicating hypersensitivity to the precursor.

Activating mu-opioid receptors in the lateral parabrachial nucleus increases c-Fos expression in forebrain areas associated with caloric regulation, reward and cognition.

The pontine parabrachial nucleus (PBN) has been implicated in the modulation of ingestion and contains high levels of mu-opioid receptors (MOPRs). In previous work, stimulating MOPRs by infusing the highly selective MOPR agonist [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) into the lateral parabrachial region (LPBN) increased food intake. The highly selective MOPR antagonist d-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) prevented the hyperphagic action of DAMGO. The present experiments aimed to analyze both the pattern of neural activation and the underlying cellular processes associated with MOPR activation in the LPBN. Male Sprague-Dawley rats received a unilateral microinfusion of a nearly maximal hyperphagic dose of DAMGO into the LPBN. We then determined the level of c-Fos immunoreactivity in regions throughout the brain. MOPR activation in the LPBN increased c-Fos in the LPBN and in the nucleus accumbens, hypothalamic arcuate nucleus, paraventricular nucleus of the thalamus and hippocampus. Pretreatment with CTAP prevented the increase in c-Fos translation in each of these areas. CTAP also prevented the coupling of MOPRs to their G-proteins which was measured by [(35)S] guanosine 5'-O-[gamma-thio]triphosphate ([(35)S]GTPgammaS) autoradiography. Together, these data strongly suggest that increasing the coupling of MOPRs to their G-proteins in the LPBN disinhibits parabrachial neurons which subsequently leads to excitation of neurons in regions associated with caloric regulation, ingestive reward and cognitive processes in feeding.

The serotonergic 5-HT(2C) agonist m-chlorophenylpiperazine increases weight-supported locomotion without development of tolerance in rats with spinal transections.

The direct serotonergic agonist, m-chlorophenylpiperazine (m-CPP), displays high efficacy at 5-HT(2C) receptors. Systemic administration of m-CPP increased dramatically the percentage of weight-supported steps made on a treadmill by rats with complete midthoracic spinal transections. The improvement in motor function occurred in rats with grafts of fetal spinal cord into the site of transection (transplant rats) and in spinal rats without grafts (spinal rats). m-CPP produced a therapeutic action with its first administration and after 14 single daily injections. In contrast, the serotonin and norepinephrine reuptake inhibitor, chlorimipramine (CMI), failed to enhance weight support during 21 days of treatment. The results imply that stimulating directly 5-HT(2C) receptors restores postural support after spinal injury. Thus, 5-HT(2C) agonists are candidates for treating spinal patients chronically without the development of tolerance.

Effects of prenatal exposure to cocaine on the developing brain: anatomical, chemical, physiological and behavioral consequences.

Earlier studies of human infants and studies employing animal models had indicated that prenatal exposure to cocaine produced developmental changes in the behavior of the offspring. The present paper reports on the results obtained in a rabbit model of in utero exposure to cocaine using intravenous injections (4 mg/kg, twice daily) that mimic the pharmacokinetics of crack cocaine in humans. At this dose, cocaine had no effect on the body weight gain of dams, time to delivery, litter size and body weight or other physical characteristics of the offspring. In spite of an otherwise normal appearance, cocaine-exposed neonates displayed a permanent impairment in signal transduction via the D1 dopamine receptor in caudate nucleus, frontal cortex and cingulate cortex due to an uncoupling of the receptor from its associated Gs protein. This uncoupling in the caudate nucleus was shown to have behavioral consequences in that young or adult rabbits, exposed to cocaine in utero, failed to demonstrate amphetamine-elicited motor responses normally seen after activation of D1 receptors in the caudate. The cocaine progeny also demonstrated permanent morphological abnormalities in the anterior cingulate cortex due to uncoupling of the D1 receptor and the consequent inability of dopamine to regulate neurite outgrowth during neuronal development. Consistent with the known functions of the anterior cingulate cortex, adult cocaine progeny demonstrated deficits in attentional processes. This was reflected by impairment in discrimination learning during classical conditioning that was due to an inability to ignore salient stimuli even when these were not relevant to the task. The impairment in discrimination learning also occurred in an instrumental avoidance task and could be shown to be due to an impairment of cingulothalamic learning-related neuronal coding. It was proposed that the selective loss of D1-related neurotransmission in the anterior cingulate cortex prevented an appropriate activation of GABA neurons and thus a loss of inhibitory regulation that is necessary for processes involved in associative attention. Taken together, these findings suggest that the uncoupling of the D1 receptor from its G protein may be the fundamental source of the anatomic, cognitive and motor disturbances seen in rabbits exposed to cocaine in utero. Moreover, the long-term cognitive and motor deficits observed in the rabbit model are in agreement with the recent reports indicating that persistent attentional and other behavioral deficits may be evident in cocaine-exposed children as they grow older and are challenged to master more complex cognitive tasks.

Direct agonists for serotonin receptors enhance locomotor function in rats that received neural transplants after neonatal spinal transection.

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.

Blockade of GABAA receptors in the medial ventral pallidum elicits feeding in satiated rats.

gamma-aminobutyric acid (GABA)-containing fibers from the nucleus accumbens shell (AcbSh) terminate in the medial ventral pallidum (VPm) and neurons in the VPm project to the lateral hypothalamus (LH). Therefore, the VPm is anatomically interposed between the AcbSh and LH, two functionally related brain regions that mediate food intake. The present study demonstrates that blockade of GABAA receptors in the VPm by local administration of bicuculline greatly increases food intake in satiated rats. The data suggest that an AcbSh-VPm-LH circuit may be involved in the control of feeding behavior.

Infusion of the serotonin1B (5-HT1B) agonist CP-93,129 into the parabrachial nucleus potently and selectively reduces food intake in rats.

Unilateral infusion of the selective 5-HT1B agonist, CP-93,129 (3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo[3,2-b]pyrid-5-one) into the parabrachial nucleus (PBN) of the pons reduced food consumption by rats. The hypophagia was dose-related (ED50 approximately 1 nmol) and associated with fewer observations of feeding and more periods of inactivity. Water intake, grooming and exploratory activity were unaffected. CP-93,129 also decreased food intake when injected into the hypothalamic paraventricular nucleus, but this action was 50-fold less potent than administration into the PBN. Autoradiography demonstrated 5-HT1B sites in the PBN; this binding was displaced by CP-93,129. The results implicate parabrachial 5-HT1B receptors in mediating serotonergic enhancement of satiation.

Effects of LSD, ritanserin, 8-OH-DPAT, and lisuride on classical conditioning in the rabbit.

d-Lysergic acid diethylamide (LSD), an agonist at the 5-HT(2A/2C) and 5-HT1A receptors, has previously been demonstrated to enhance associative learning as measured by accelerated acquisition of the rabbit's classically conditioned nictitating membrane (NM) response. The present study examined further the role of these receptors in the action of LSD. LSD (30 nmol/kg, I.V.) significantly enhanced conditioned response (CR) acquisition to both tone and light conditioned stimuli (CSs), while the 5-HT1A receptor agonists 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT; 50 and 200 nmol/kg) and lisuride (0.3-30 nmol/kg) had no effect. Ritanserin (6.7-6700 nmol/kg, S.C.), a selective 5-HT(2A/2C) receptor antagonist, retarded acquisition of CRs to both tone and light CSs in a dose-dependent manner. Ritanserin (6.7-670 nmol/kg, S.C.) also dose dependently antagonized the enhancement of CR conditioning produced by LSD (30 nmol/kg, I.V.) to both tone and light CSs. We conclude that the enhancement of CR acquisition by LSD was due to an action at the 5-HT(2A/2C) receptor. These results suggest that the 5-HT(2A/2C) receptor plays an important role in learning.

New evidence for neurotransmitter influences on brain development.

The early appearance of monoamine systems in the developing mammalian CNS suggests that they play a role in neural development. We review data from two model systems that provide compelling new evidence of this role. In one model system-in utero exposure to cocaine-specific and robust alterations are seen in dopamine-rich areas of the cerebral cortex, such as the anterior cingulate cortex: D1 receptor-G protein coupling is greatly reduced, the GABAergic system is altered and pyramidal dendrites undergo excessive growth. In a second model system-a transgenic mouse line in which the gene that encodes monoamine oxidase A (MAOA) is disrupted, resulting in excessively high 5-HT levels-barrels fail to form in the developing somatosensory cortex. Both models reveal the effects of very early manipulation of monoamines on forebrain development, and the long-term anomalies that persist into adulthood.

CP-94, 253: a selective serotonin1B (5-HT1B) agonist that promotes satiety.

The selective 5-HT1B agonist CP-94,253 (3- (1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3, 2-b] pyridine) (5-40 mumol/kg) reduced the intake of both pellets and a 10% solution of sucrose (ID50 = 12.5 and 22.8 mumol/kg, respectively) in mildly deprived rats. Time-sampled observations revealed that CP-94,253 terminated feeding earlier, without disrupting the continuity of feeding. CP-94,253 increased standing but did not promote resting during satiation. Microstructural analysis of licking indicated that CP-94,253 decreased the frequency, but not the size, of bursts and clusters of licks without altering oral motor efficiency. The peripherally acting 5-HT1B agonist, CP-93,129 (3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one) had no effect on food intake. These results imply that CP-94,253 probes a role for central 5-HT1B receptors in the regulation of meal size and duration, but that recruitment of other 5-HT receptor subtypes may be needed for the full expression of satiety.

d-fenfluramine anorexia: dissociation of ingestion rate, meal duration, and meal size effects.

In the present study, we ask whether the suppressive effect of d-fenfluramine (d-FEN) on short-term intake can be better explained in terms of a primary action on particular behavioral parameters (e.g., ingestion rate or meal duration), as proposed by several investigators, or in terms of a primary effect on an intake "target" that can be achieved via diverse behavioral strategies. We applied two specialized intake testing paradigms that constrain the behavioral structure of the rat's meal in different ways, and determined whether or not the meal-size result varied in turn. (1) In the intraoral intake test, the rate of ingestion was clamped by the rate (1.0 ml/min) at which the test stimulus (12.5% glucose) was intraorally delivered. A d-FEN (3 mg/kg) suppression of intraoral intake was obtained demonstrating that ingestion rate adjustment is not necessary for the anorexic effect. In addition, for both d-FEN and vehicle conditions, comparable amounts were consumed when the intraoral intake test was either continuous or interrupted for 10 min beginning 6 min after test onset. For d-FEN, the increase in meal duration (mean = 11.98 min) required to compensate for the imposed interruption indicates that the drug does not specify an absolute limit for meal duration. (2) In the drop size-controlled spout-licking test, the volume of 12.5% glucose delivered for each lick was fixed at either 8 or 4 microliters. There was an overall reduction in intake with d-FEN (0.75 mg/kg), but as under vehicle injection conditions, the number of licks emitted approximately doubled when lick volume was halved. As a result, meal size was unaffected by the drop size manipulation. The drop size manipulation affected several other behavioral parameters under respective d-FEN and vehicle injection conditions, including: average rate of ingestion (ml/min), initial ingestion rate, and ingestion duration (meal duration minus pause time). The two experiments together demonstrate that the anorexic effect of d-FEN does not depend on adjustment of any particular behavioral parameter. The results suggest, rather, that given doses of d-FEN establish a particular degree of intake suppression that the rat defends via diverse behavioral strategies.

Serotonergic control of the organization of feeding and satiety.

Studies in which serotonergic drugs were administered either systemically or directly into central sites have implicated 5-HT in the inhibitory control of feeding in mammals. In animal models and in humans, 5-HT agonists such as fenfluramine, fluoxetine and sertraline reduced the rate of eating and the size of meals in a manner suggesting that increasing serotonergic neurotransmission specifically enhanced satiation. In rodents, directly acting agonists at 5-HT1B, 5-HT2C or 5-HT2A receptors decreased food intake but by different behavioral mechanisms. Stimulation of the 1B and 2C subtypes may probe physiological roles in feeding and satiety. The former receptors may be involved primarily in regulating meal size and the latter more in controlling eating rate. Activation of both may be required for complete expression of behavioral satiety. By contrast, stimulating 2A sites may simply disrupt the continuity of feeding. Drugs that stimulate 5-HT1A autoreceptors increase food intake, presumably by acutely reducing the firing of serotonergic neurons in the brain. The hypothalamic paraventricular nucleus (PVN) has been proposed as an important terminal field in the forebrain that is involved in 5-HT's satiety role although recent studies implicate extra-PVN regions in this function. Peripherally administered 5-HT also decreases food intake in rats in a behaviorally specific manner. Studies with antagonists and with structural analogs of 5-HT revealed that 5-HT's peripheral satiety action involves 5-HT1-like and 5-HT2-like mechanisms. Thus, within and outside the brain, multiple pharmacological and behavioral mechanisms contribute to serotonergic functions in ingestion. The rich body of data from preclinical investigation in animals provides the foundation for therapeutic development in humans.

Prenatal exposure to cocaine selectively disrupts motor responding to D-amphetamine in young and mature rabbits.

Acute administration of D-amphetamine probed the functional effects of prenatal exposure to cocaine on the integrity of monoaminergic systems in preweanling (48-56 days old) and adult (> or = 140 days old) Dutch belted rabbits. D-Amphetamine sulfate (0, 0.3, 1.0, 3.3 and 6.0 mg/kg, s.c.) produced equivalent dose-related reductions in food intake in 180 day-old rabbits that had been exposed in utero on gestational days 8-29 to cocaine or saline. Intrauterine exposure to cocaine also did not alter the incidence of exploratory behaviors stimulated by D-amphetamine during the anorexia test. In contrast, however, prenatal cocaine virtually eliminated stereotyped head bobbing elicited by the highest dose of D-amphetamine. When responses to 5.0 mg/kg D-amphetamine were measured during a 90-min open field test, prenatal cocaine prevented head bobbing in preweanling rabbits and reduced this behavior by 92% in 140 day-old adults. Prenatal cocaine also diminished the intensity of other motor responses in the open field in the adults but not in preweanlings. In normal rabbits, the D1 antagonist R(+)-SCH 23390 (0.01 mg/kg, s.c.) blocked D-amphetamine-induced head bobbing. Thus, prenatal exposure to cocaine produces an early and persistent deficit in behavioral responding to a high dose of D-amphetamine. The deficit is especially selective at the time of weaning, broadens to affect more motor behaviors with maturation and may reveal impaired D,-mediated dopaminergic neurotransmission in the brain.

Lesions of area postrema attenuate but do not prevent anorectic action of peripheral serotonin in rats.

These studies assessed the effect of selectively ablating the area postrema (AP) on the action of peripheral serotonin [5-hydroxytryptamine (5-HT)] to reduce food intake in rats. Intraperitoneal 5-HT (0, 2.0, 4.0, and 8.0 mumol/kg) reduced the intakes of sweetened mash during a 30-min test in controls (APC) and in AP-lesioned rats (APX). The anorexia was dose dependent in controls but the dose-response function was flat after AP lesions. In another study, 2.0 mumol/kg 5-HT reduced intakes of both groups by approximately 25%, but AP lesions blunted the effect at 8.0 mumol/kg 5-HT (APX, -30% vs. APC, -85%). Behavioral analysis revealed that, compared with controls, AP lesions eliminated the decrease in frequency of feeding and reduced the incidence of resting and of an aberrant posture observed after 8.0 mumol/kg. Thus peripheral 5-HT decreases food intake in rats with AP lesions. Multiple mechanisms appear to be involved in the ability of peripheral 5-HT to reduce feeding. A high dose of 5-HT promotes responses associated with satiation but also produces behavioral toxicity; these effects involve the AP. Lower doses appear to engage processes that do not rely on the function of this circumventricular organ.

Intrauterine exposure to cocaine produces a modality-specific acceleration of classical conditioning in adult rabbits.

Previous studies had demonstrated that in utero exposure to cocaine produces structural changes in the development of the rabbit's anterior cingulate cortex. Because the anterior cingulate cortex has been proposed to subserve a variety of cognitive processes including associative learning, we investigated the effects of intrauterine exposure to cocaine on the acquisition of the rabbit's classically conditioned nictitating membrane response. Adult, sexually mature rabbits born of dams that had received intravenous injections of either saline or cocaine (4 mg/kg, twice a day) from day 8 to day 29 of gestation were classically conditioned by pairing tone and light CSs with an airpuff US. Rabbits that had been exposed to cocaine in utero demonstrated a more rapid acquisition of CRs to a tone CS but not to a light CS as compared with saline controls. Control experiments indicated that the accelerated learning to the tone CS was not due to sensitization, pseudoconditioning, altered baseline rate of responding, an increased responsiveness to the airpuff US, or to a change in the intensity threshold of the tone CS for elicitation of CRs. We conclude that in utero exposure to cocaine alters the processing of auditory stimuli and this leads to an abnormally rapid acquisition of CRs. It is suggested that this functional consequence of prenatal exposure to cocaine is due to structural abnormalities in anterior cingulate cortex.

Serotonin contracts the isolated rat pylorus via a 5-HT2-like receptor.

Serotonin (5-HT) produced concentration-dependent contractions of the isolated rat pylorus in vitro. Serotonergic contractions were antagonized by the calcium L-channel blocker, diltiazem, but not by tetrodotoxin or atropine. Three drugs that block 5-HT2 receptors, ketanserin, xylamidine, and methysergide, unsurmountably inhibited contractions to 5-HT. In contrast, antagonists of 5-HT3, 5-HT1A/1B, beta-adrenergic, or alpha 1-adrenergic receptors did not alter the response to 5-HT. Devazepide, an antagonist of cholecystokinin (CCK) type-A receptors, blocked contraction produced by CCK-8 but not by 5-HT. Conversely, the 5-HT2 antagonists did not affect CCK-stimulated contraction. These results suggest that 5-HT contracts the pylorus by a 5-HT2-like receptor on muscle and that this response occurs independently of CCKergic receptor mechanisms. Furthermore, the parallel between the overall pharmacological profile for serotonergic contraction of the pylorus and that observed previously for the anorectic action of peripheral 5-HT makes the pylorus a logical candidate for peripheral serotonergic control of feeding.

Serotonin-induced muscle contraction in rat stomach fundus is mediated by a G alpha z-like guanine nucleotide binding protein.

Serotonin (5-HT) potently contracts the fundus of the rat stomach; however, the associated transduction pathway has not been described fully. Experiments were performed in an attempt to gain insight into the coupling mechanism associated with this fundal 5-HT receptor. 5-HT-stimulated [35S]GTP gamma S binding to a protein which was recognized by anti-G alpha Z antiserum in a Mg(++)-dependent fashion. 5-HT increased [35S]GTP gamma S binding in the fundus, but not in the corpus of the rat stomach. 5-HT also enhanced the binding of [alpha-32P]GTP to the fundal protein and increased the hydrolysis of GTP to GDP in fundal membranes. The fundal protein which binds GTP is 25 to 29 kDa in size whereas the brain G alpha Z protein which is recognized by the anti-G alpha Z antibody is a 41 kDa protein. Mixing experiments revealed that the fundal guanine nucleotide binding protein does not appear to be a proteolytic product of the 41 kDa G alpha Z protein. Activating protein kinase C with phorbol-12-myristate, 13-acetate induced a concentration-dependent, noncompetitive inhibition of [35S]GTP gamma S binding to the fundal protein, and of 5-HT-induced contraction of fundal strips. Phorbol-12-myristate, 13-acetate did not alter carbachol- or KCl-mediated fundus contraction. Furthermore, the activation of [35S]GTP gamma S binding by serotonergic agonists and its inhibition by pharmacological antagonists corresponded to the known actions of these agents on contraction of fundal muscle. The results provide evidence that the 5-HT receptor in the rat stomach fundus is coupled directly or indirectly to a G alpha z-like protein which may mediate 5-HT-induced contraction in this tissue.