Effects of serotonergic and cholinergic antagonists on suckling behavior of neonatal, infant, and weanling rat pups

In Experiment 1, Sprague-Dawley rat pups at postnatal days 3-4, 7-8, 10-11, 15-16, and 23-24 were tested for suckling behavior on their anesthesized multiparous dams following administration of metergoline or scopolamine. The serotonergic antagonist, metergoline, inhibited suckling in 3- to 4- and 7- to 8-day-old rat pups, but was not found to influence suckling in older pups. Scopolamine, a cholinergic antagonist, reduced suckling primarily in 3- to 4-day-old pups. In Experiment 2, parity of the maternal female was manipulated to assess whether this variable would influence suckling behavior and the effects of metergoline on suckling of weanlings. Baseline levels of suckling were reduced in 23- to 24-day-old pups of primiparous dams when compared with multiparous-derived offspring. Administration of metergoline increased the amount of time that these primiparous-derived pups spent attached to nipples, but did not influence suckling of offspring of multiparous dams, perhaps as a result of a ceiling effect on suckling behavior in these animals.     

Drug and environmentally induced manipulations of the opiate and serotonergic systems alter nociception in neonatal rat pups

The influence of drug- and environmentally induced alterations in serotonergic and opiate activity on pain sensitivity was assessed in 6-day-old Sprague-Dawley-derived rat pups using tail flick-testing procedures. The opiate agonist morphine was observed to induce tail flick analgesia that was blocked by concurrent administration of the opiate antagonist naloxone. Similarly, the serotonergic agonist quipazine induced analgesia that was blocked by pretreatment with the serotonergic antagonist metergoline. Naloxone alone did not alter tail flick responsivity in non-isolated, nondeprived neonates, suggesting that the opiate system may not exert a significant tonic inhibition of pain sensitivity in neonates. In contrast, the serotonergic system may exert some tonic analgesic influence at this age, given that metergoline was observed to induce slight hyperalgesia in nondeprived, non-isolated neonates. Twenty four hours of food and maternal deprivation, shown previously to increase brain serotonin and 5-hydroxyindole acetic acid and their ratio in neonates (L. P. Spear & F. M. Scalzo, 1984, Developmental Brain Research, in press) was observed to induce tail flick analgesia, an effect blocked by metergoline. Isolation from siblings and the dam and nest for 30 min also induced tail flick analgesia; this analgesia was blocked by treatment with naloxone prior to testing. Together, these experiments support the suggestion that the serotonergic and opiate systems may regulate pain sensitivity even in neonatal rat pups, with agonist- or environmentally precipitated increases in serotonergic or opiate activity inducing significant analgesia during the early postnatal period.     

Serotonin's influence on predatory behavior of highly aggressive cba and weakly aggressive DD strains of mice

The effects of serotonin were studied on locust-killing behavior of mice from low (DD) and high (CBA) predatory aggressive strains. 5-HTP injected intraperitoneally (50 and 100 mg/kg) or 5-HT administered into the lateral ventricle (10 μg) significantly reduced locust-killing behavior in highly aggressive CBA mice. Imipramine (20, 30, and 40 mg/kg) elicited a dose-dependent inhibitory effect on predatory behavior. Fluoxetine (10 and 20 mg/kg) alone had a slight influence on locust-killing behavior but potentiated the action of the subthreshold dose of 5-HTP (25 mg/kg). Pretreatment with the blocker of 5-HT₂ type receptors methysergide (2 mg/kg) abolished the inhibitory effect of 5-HTP. These finding indicate that serotonin of the brain exerts an inhibitory effect on predatory behavior in mice. In contrast, neither lesion of the dorsal raphe nucleus (although significantly depleting the brain serotonin) nor treatment with methysergide (2 mg/kg) induced locust-killing behavior in weakly aggressive DD mice. Low predatory aggressiveness in DD mice is suggested to be related to the low tonus of the mechanisms activating killing behavior rather than to excessive serotonergic inhibitory influences.     

The medial amygdala: Serotonergic inhibition of shock-lnduced aggression and pain sensitivity in rats

Two aspects of the amygdaloid complex (corticomedial and basolateral) were examined with reference to serotonergic inhibition of shock-induced aggression. Fighting was significantly depressed by serotonergic stimulation (5-HT, 10 μg bilateral) in the corticomedial amygdala while serotonergic blockade (methysergide, 5 μg bilateral) in this region increased levels of fighting. No consistent effects were obtained with serotonergic manipulation of the basolateral amygdala. Further investigation revealed that the state of serotonergic activity in medial amygdaloid sites was associated with concomitant alterations in the animals' sensitivity to footshock. Results are discussed in relation to a) a general inhibitory role of serotonin in behavioural mechanisms and b) a dopaminergic-serotonergic balance for behavioural arousal involving medial amygdaloid nuclei.     

Modulation of baseline behavior in rats by putative serotonergic agents in three ethoexperimental paradigms

The present study adopts an ethoexperimental approach to examine the deportment subsequent to alteration in serotonin (5-HT) neurotransmission following treatment with site-specific neuropharmacological probes. The impact of perturbation in (5-HT) neurotransmission on baseline behavior was analyzed employing three animal models of anxiety, i.e., hole-board, elevated plus maze, and bright/dark arena. Inbred male rats (Wistar strain, weighing between 150 and 200 g) were used in this study. The vivarium and the behavioral laboratory were specially designed to permit operation of reversed light-dark cycle and all experiments were performed during the dark period. Pharmacological tools selected to influence 5-HT levels include (1) a combination of tranylcypromine and tryptophan (TCP + TRYPT) (0.75 mg/kg + 40 mg/kg) which augments 5-HT biosynthesis; (2) p-chlorophenylalanine (PCPA: 200 mg/kg), an inhibitor of 5-HT biosynthesis; and (3) 5-HT reuptake blockers, namely zimelidine (ZIM) (40 mg/kg) and fluoxetine (FLU) (10 mg/kg). Rats under the influence of PCPA exhibited anxiolytic response, whereas those under treatments to raise 5-HT levels, viz., TCP + TRYPT, ZIM and FLU, displayed anxiogenic-like reactions. Several other agents known to specifically interact with 5-HT receptor subtypes were also tested. 5-HT2 receptor stimulants, such as quipazine (5 mg/kg) and MK 212 (0.5 mg/kg), were found to be anxiogenic. Buspirone (2 mg/kg), a 5-HT1 agonist, surmounted normal behavioral inhibition. However, another 5-HT1 stimulant, 8-OH-DPAT (0.025 mg/kg), had anxiogenic action. Pretreatment with 5-HT3 antagonists [zacopride (2 mg/kg) and GR 38032F (0.1 mg/kg)] and putative 5-HT1 antagonist [propranolol (10 mg/kg)] resulted in borderline disinhibition of normal behavioral inhibition to novel environments. In contrast, cyproheptadine (0.5 mg/kg), a 5-HT2 antagonist, provoked anxiogenic-like behavior. Altogether, uniform results were obtained for each probe in all the three models, suggesting that the battery of anxiety tests chosen in this study is reliable and sensitive to detect unknown pharmacological responses. The results support the hypothesis that stimulation of serotonergic neurotransmission heightens normal anxiety, whereas its blockade releases normal behavioral inhibition. Furthermore, this work establishes the validity of using the three paradigms in evaluating the involvement of multiple neurotransmitter receptors in the control of behavior of rodents under natural circumstances and also detects any aberration following exposure to novelty and stress.     

Decline of maternal behavior in the virgin and lactating rat.

Sensitized virgins and postpartum lactating mothers, both exhibiting maternal behavior, were given donor litters that increased in age by 1 day, for 28 days, starting at the onset of maternal behavior. Each day females were tested for maternal behavior with pups 4-8 days old: Maternal care (i.e., nursing/crouching, retrieving, nest building and licking) and maternal withdrawal, rejection, and prevention of nursing were recorded. After the ninth day, females were also tested with the progressively older pups from 10 to 28 days of age with which they were living. Virgins and lactating mothers showed generally similar patterns of maternal care although some differences were found, and they declined in maternal behavior toward the older pups in a similar manner. Maternal behavior did not decline in tests with younger pups. The results are interpreted as supporting the hypothesis that the decline as well as the maintenance of maternal behavior postpartum is nonhormonally mediated.     

Differential effects of a 5-HT2 receptor blocker on alcohol intake in rats selectively bred for high and low catecholamine responses to stress

The effect of the selective 5-HT2 receptor blocker ritanserin on alcohol consumption was investigated in two strains of rats selectively bred for high and low catecholamine responses to stress. Rats were forced to drink a 5% alcohol solution for 10 days. For the subsequent six days, animals were injected subcutaneously with 2.5 mg/kg/2 ml ritanserin or vehicle only, and both a 5% solution of alcohol and water were presented to the animals. Ritanserin affected neither water nor total fluid intake. Furthermore, no effect of ritanserin on alcohol consumption could be demonstrated in high-responding rats, whereas in low-responding rats a very pronounced ritanserin-induced reduction in alcohol intake was observed. Results are discussed in terms of mediating effects of serotonergic neurons on mesolimbic dopaminergic reward systems related to drug addiction.     

Evidence for a possible functional interaction between serotonergic and cholinergic mechanisms in memory retrieval

A total of three experiments were conducted. In Experiment 1, the dose-dependent effects of the pretest administration of the serotonergic agonist alaproclate and the selective muscarinic cholinergic agonist oxotremorine, alone and in combination, were assessed in a one-trial inhibitory avoidance task. A clear dose-dependent enhancement of performance was demonstrated as a result of all three treatment conditions, which could not be explained in terms of nonspecific effects of the drugs on behavior in general. In addition, the facilitation of retrieval performance produced by the combined treatment of alaproclate and oxotremorine was observed at dose levels well below those observed following administration of either compound alone. In Experiment 2 attempts were made to block the enhancements of retention resulting from the different treatment conditions (alaproclate, oxotremorine, or the combination of alaproclate and oxotremorine) by pretreating the mice with either scopolamine (a muscarinic cholinergic antagonist) or quipazine (a serotonergic agonist). The results of these experiments indicate that (a) quipazine completely blocked the enhancement of retrieval resulting from alaproclate but not that following oxotremorine or oxotremorine in combination with alaproclate, while (b) scopolamine blocked the enhancement of retrieval resulting from oxotremorine alone as well as that resulting from alaproclate plus oxotremorine but failed to block the memory enhancement resulting from alaproclate. The present results lend further support to the view that both serotonin and acetylcholine play important roles in memory retrieval. More importantly, the results of the present series of experiments provide additional support for a functional interaction between the serotonergic and cholinergic nervous systems in the mediation of behavior.     

Improvements in hippocampal-dependent learning and decremental attention in 5-HT(3) receptor overexpressing mice

The 5-HT3 receptor for serotonin is expressed within limbic structures and is known to modulate neurotransmitter release, suggesting that this receptor may influence learning and memory. Perturbations in serotonergic neurotransmission lead to changes in the ability to attend, learn, and remember. To examine the role of 5-HT3 receptors in learning, memory, and attention, 5-HT3 receptor overexpressing (5-HT3-OE) transgenic mice and their wild-type littermates (WT) were tested in Pavlovian contextual and cued fear conditioning, fear extinction, and latent inhibition (LI) paradigms. Prepulse inhibition (PPI) was assessed to reveal changes in sensorimotor gating. Additionally, anxious behaviors, shock sensitivity, and reactions to novel stimuli were evaluated. 5-HT3-OE mice displayed enhanced contextual conditioning, whereas cued conditioning remained the same as that of WT mice. 5-HT3-OE mice did not differ from WT in extinction rates to either the context or cue. LI was enhanced for 5-HT3-OE mice compared to WT. PPI remained unchanged. No differences in sensitivity to footshock or startle were found. However, 5-HT3-OE mice demonstrated heightened exploratory behavior in response to novel environmental stimuli and decreased anxiety as measured in the elevated plus-maze. Results indicate that overexpression of the 5-HT3 receptor in mouse forebrain results in enhanced hippocampal-dependent learning and attention. Enhanced inspective behavior in response to novelty may contribute to the observed improvements in learning, memory, and attention due to 5-HT3 receptor overexpression.     

Effects of serotonin-mimetic drugs on mouse-killing behavior

Muricidal behavior induced in rats by social isolation or by olfactory bulb ablation was blocked following IP administration of serotonin (5–HT) agonist 8-OH-DPAT and 5-MeODM and by 5-HT uptake inhibitors, fluoxetine and indalpine. Among uptake inhibitors, although fluoxetine has a higher IC₅₀ and a higher Ki, it is apparently more efficient than indalpine. The 5-HT agonist, 8-OH-DPAT, acting at a putative 5-HT_1A receptor, appears more efficient on muricidal inhibition than 5-MeODM, at a much lower dosage. It is highly probable that 5-HT_1A receptor rather than 5-HT_1B is involved in the antimuricidal effect of serotonin-mimetic drugs. Since 5-HT mimetic drugs blocked mouse-killing behavior of bulbectomized rats, we suggest that in the sequence of events in muricidal inhibition 5-HT circuits participate after gabaergic modulation from olfactory bulbs.     

Opiates and homing

Beginning at 15 days of age. Long-Evans rat pups were trained to run toward their home cage in a T-maze task. Morphine (.5-1.0 mg/kg sc) slowed initial acquisition running times but did not change the number of trials required to learn the position habit. Morphine markedly impeded extinction of the homing behavior. Opiate-treated animals ran as accurately and as quickly toward home on the 12th day of extinction as on the first (10 trials given per day). Conversely, naloxone (1 mg/kg sc) reduced resistance to extinction. The morphine effect was not state-dependent since the drug also impeded extinction in animals that had acquired the task under saline. The morphine effect was blocked by naloxane, which indicates that the increased resistance to extinction was due to an opiate receptor effect. These results indicate that morphine has a strong capacity to sustain a social habit in the absence of reinforcement.     

Serotonergic and opiate interactions in the modulation of drug- and environmental-induced analgesia in the neonatal rat pup

Serotonergic and opiate interactions in the modulation of drug- and environmental-induced analgesia were assessed in 6-day-old Sprague-Dawley-derived rat pups using tail-flick testing procedures. In these experiments the serotonergic antagonist metergoline was observed to attenuate both the analgesia induced by the opiate agonist morphine and the analgesia induced by isolation from siblings and the dam, an environmental manipulation which has previously been shown to be associated with increases in opiate activity. In contrast, the opiate antagonist naloxone was observed to be ineffective in blocking not only analgesia induced by the serotonergic agonist quipazine, but also analgesia induced by long-term deprivation from the dam and food, a manipulation that has been previously reported to induce increases in serotonergic utilization. These results suggest that in the neonate, as in the adult, the serotonergic modulation of nociception appears to occur "downstream" from the opiate systems serving to regulate nociception following both drug- and environmental-induced alterations in pain sensitivity. Analgesia induced by long-term deprivation from food and the dam appears to be strongly related to increases in serotonergic activity and relatively unaffected by opiate antagonism, whereas analgesia induced by isolation from siblings and the dam may be related to increases in opiate activity, but modulated by serotonergic systems serving to regulate pain responsivity. Thus alterations in the environment, mediated at least in part by alterations in opiate and serotonergic activity, appear to play an important role in influencing the sensitivity of the neonate to pain stimuli.     

Multiple serotonergic mechanisms contributing to sensitization in aplysia: evidence of diverse serotonin receptor subtypes

The neurotransmitter serotonin (5-HT) plays an important role in memory encoding in Aplysia. Early evidence showed that during sensitization, 5-HT activates a cyclic AMP-protein kinase A (cAMP-PKA)-dependent pathway within specific sensory neurons (SNs), which increases their excitability and facilitates synaptic transmission onto their follower motor neurons (MNs). However, recent data suggest that serotonergic modulation during sensitization is more complex and diverse. The neuronal circuits mediating defensive reflexes contain a number of interneurons that respond to 5-HT in ways opposite to those of the SNs, showing a decrease in excitability and/or synaptic depression. Moreover, in addition to acting through a cAMP-PKA pathway within SNs, 5-HT is also capable of activating a variety of other protein kinases such as protein kinase C, extracellular signal-regulated kinases, and tyrosine kinases. This diversity of 5-HT responses during sensitization suggests the presence of multiple 5-HT receptor subtypes within the Aplysia central nervous system. Four 5-HT receptors have been cloned and characterized to date. Although several others probably remain to be characterized in molecular terms, especially the Gs-coupled 5-HT receptor capable of activating cAMP-PKA pathways, the multiplicity of serotonergic mechanisms recruited into action during learning in Aplysia can now be addressed from a molecular point of view.     

Ritanserin and voluntary alcohol intake in rats

In a previous study (Rammsayer & Vogel, 1991), rats selectively bred for high and low catecholamine responses to stress showed a selective response to the 5-HT2 receptor blocker ritanserin. However, it remained unclear whether selective breeding resulted in a decrease in 5-HT responsivity, as suggested by the lack of an effect in high stress responding rats, or in an increase in 5-HT responsivity, as suggested by ritanserin-induced reduction in alcohol intake in low-responding rats. To answer this question, nonselectively bred rats were forced to drink a 5% alcohol solution for 10 days. For the subsequent six days, animals were injected subcutaneously with 2.5 mg/kg/2 ml ritanserin or vehicle only, and both a 5% solution of alcohol and water were presented to the animals. Ritanserin neither affected alcohol nor total fluid intake suggesting that in the general population of N/NIH (Hansen) rats as well as in rats of the same strain selectively bred for high catecholamine responses, mesolimbic dopaminergic activity is not effectively modulated by specific blockade of 5-HT2 receptors. However, a very pronounced ritanserin induced difference in daily water intake between nonbred male and female rats became evident.     

Rapid visual learning in the rat: Effects at the 5-HT 1a receptor subtype

The 5-hydroxytryptamine 1a (5-HT 1a ) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 0.15 mg/kg) impaired rats' rapid visual learning on a computerized maze. This treatment also increased decision time (DT) but the learning impairment was not necessarily a side-effect of slower responding because, in this task, responses made at long DT are more accurate than those at short DT. The selective 5-HT 1a receptor antagonist WAY-100635 (0.3 mg/kg) was itself without effect on accuracy, but was effective in reversing effects of 8-OH-DPAT (on both accuracy and DT). Within problems (i.e., over the 40-60 trials of a single discrimination), performance was reduced by treatment with 8-OH-DPAT at all stages of learning. We conclude that this effect is mediated through the 5-HT 1a receptor site (rather than through some other serotonergic receptor site or non-specific mechanism) as it was reversible by treatment with WAY-100635. Although it could still arise from behaviourally non-specific effects, the performance deficit finds its best account in terms of the psychological processes necessary to visual learning. Its reversal with WAY-100635 offers support to the hypothesis that 5-HT 1a receptor antagonists could improve cognitive function, under conditions of pre-existing impairment due to overactive serotonergic inhibition, as is thought to occur in Alzheimer's disease.     

Taste aversions to mother's milk: the age-related role of nursing in acquisition and expression of a learned association.

The development of taste aversion learning to novel cues contained in mother's milk was examined in rat pups. Pups receiving distinctive mild by experimenter-delivered oral infusions followed by toxicosis formed an aversion to the dam's diet. Robust aversions were learned as early as Day 10 and were retained for at least 11 days. When the same distinctive milk was obtained directly from a foster mother through nursing, however, only weanling-age pups (over 20 days) formed an aversion. X-ray analysis of nipple location in the mouths of suckling pups suggested that pups between the ages of 10 and 21 days receive milk at a similar tongue locus. Flavored milk was then delivered at specific time intervals in controlled quantities through tongue cannulas implanted at loci corresponding to the nipple position shown by the X-rays. Cannulated preweanling pups that were attached to a nipple during milk delivery failed to associate the taste cue with illness, whereas both preweanlings off the nipple and weanlings on the nipple acquired aversions to the taste cue in the milk. The evidence obtained in these experiments suggests that pups of all ages are incapable of expressing a taste aversion in a nursing situation and that preweanling pups in particular are also dificient in acquiring aversions within a suckling context. The inability of preweanling pups to acquire taste aversions in a nursing situation appears to result from a failure to associate taste cues with illness rather than a failure to detect taste cues obtained from a nipple.     

New lessons from knockout mice: The role of serotonin during development and its possible contribution to the origins of neuropsychiatric disorders

Serotonin (5-HT) modulates numerous processes in the central nervous system that are relevant to neuropsychiatric function and dysfunction. It exerts significant effects on anxiety, mood, impulsivity, sleep, ingestive behavior, reward systems, and psychosis. Serotonergic dysfunction has been implicated in several psychiatric conditions but efforts to more clearly understand the mechanisms of this influence have been hampered by the complexity of this system at the receptor level. There are at least 14 distinct receptors that mediate the effects of 5-HT as well as several enzymes that control its synthesis and metabolism. Pharmacologic agents that target specific receptors have provided clues regarding the function of these receptors in the human brain. 5-HT is also an important modulator of neural development and several groups have employed a genetic strategy relevant to behavior. Several inactivation mutations of specific 5-HT receptors have been generated producing interesting behavioral phenotypes related to anxiety, depression, drug abuse, psychosis, and cognition. In many cases, knockout mice have been used to confirm what has already been suspected based on pharmacologic studies. In other instances, mutations have demonstrated new functions of serotonergic genes in development and behavior.     

Injection of 5,7-dihydroxytryptamine into the B3 raphe region of neonatal rat pups induces hyperalgesia but only slight alterations in ingestion-related behaviors.

The effects of intrabrainstem injections of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the B3 raphe region (nucleus raphe magnus and nucleus reticularis paragigantocellularis) on early ingestive behavior and nociception were assessed in Sprague-Dawley rat pups during the first postnatal week. Lesions resulted in a marked depletion of serotonin (5HT) in hindbrain without influencing 5HT levels in forebrain. Pretreatment with desipramine (DMI) resulted in a sparing of noradrenergic neurons from neurotoxic effects. The B3 lesion resulted in significant hyperalgesia as reflected by decreased latencies in tail flick testing. Although nipple attachment latencies in suckling tests were slightly increased by the lesion, no notable effects on mouthing or other ingestive-related behaviors were observed in testing conducted in an independent ingestion paradigm. These results suggest that whereas B3 serotonergic neurons may be functioning in an adult-typical manner to regulate analgesia during the early postnatal period, this raphe region may play only a slight role in the modulation of ingestion-related behaviors early in life.     

Disruption of noradrenergic, but not serotonergic or opiate, functioning blocks both cardiac and behavioral components of the orienting response in preweanling rats

Previous work in this laboratory established that selective attention, as measured by the behavioral and autonomic expressions of the orienting response (OR), is not disrupted by either dopaminergic or cholinergic receptor blockade. The present experiments extended this pharmacological analysis of the OR. In Experiment 1, preweanling rats were injected with methysergide maleate, a serotonin receptor blocker. Neither the behavioral nor the heart rate (HR) component of the OR was attenuated. In Experiment 2, the opiate receptor blocker naltrexone also failed to inhibit the HR and behavioral expressions of the OR. alpha-1 adrenergic receptor blockade with WB-4101 in Experiment 3 abolished both the HR and behavioral ORs to the pulsating tone. In Experiment 4, clonidine, which inhibits release of norepinephrine by stimulating alpha-2 autoreceptors, attenuated both behavioral and HR ORs to the pulsating tone in a dose-dependent manner. These data, in combination with the prior findings, suggest that norepinephrine is critically involved in the central process underlying the OR in the rat. Dopaminergic, cholinergic, serotonergic, and opiate receptor blockades do not impair selective attention as indexed by HR and behavioral ORs to an auditory stimulus. In contrast, disruption of noradrenergic functioning via either alpha-1 receptor blockade or alpha-2 receptor stimulation disrupts both the HR and behavioral components of the OR. These results indicate that integrity of central noradrenergic functioning is essential for expression of the OR and for stimulus-directed attention.