Evidence of deprenyl-insensitive apoptosis of nigral dopamine neurons during development.

Apoptosis of dopamine neurons occurs naturally in the substantia nigra during development, culminating in approximately 30% loss of these cells during the perinatal period. Deprenyl, independent of its monoamine oxidase (MAO)-B inhibitory properties, can prevent dopamine neuronal apoptosis in models of neurodegeneration. Our current study demonstrate that apoptotic death of dopamine neurons during development is insensitive to daily treatment of pregnant mothers and then newborns with deprenyl (0.1, 1, or 10 mg/kg). This result is not due to poor crossing of the placental and blood-brain barriers, since deprenyl caused a dose-dependent inhibition of brain MAO-B activity in pups at birth. Determining the pathway(s) leading to deprenyl-insensitive apoptosis of nigral dopamine neurons in development may shed light on mechanisms underlying the premature death of dopamine neurons in neurodegenerative disorders.

Activation of adenosine A1 receptors in the nucleus accumbens impairs inhibitory avoidance memory.

Potent and highly selective adenosine A1 and A2 receptor agonists were bilaterally injected into the nucleus accumbens of mice 10 min prior to inhibitory avoidance training. Retention of the inhibitory avoidance response was assessed 24 h after training. Intra-ACB activation of A1 receptors, but not A2a receptor activation, significantly impaired the performance of mice during the subsequent retention test. Furthermore, the retention deficit produced by activation of A1 receptors was significantly attenuated by pretreating mice with a highly selective A1 receptor antagonist. These findings suggest that endogenous adenosine may modulate information processing in the ventral striatum via adenosine A1 receptors.

Role of adenosine A2a receptors in the nucleus accumbens.

Adult male mice were stereotaxically implanted with permanent indwelling guide cannulae for bilateral injections into the nucleus accumbens (ACB). The effects on spontaneous locomotor activity of selective agonists for adenosine receptor subtypes were examined following bilateral injections into the ACB. Intra-ACB injections of CGS 21680, a potent and selective agonist at striatal adenosine A2a receptors, elicited pronounced, dose-related reductions in locomotor activity whereas similar bilateral dosages of CPA, a selective agonist at adenosine A1 receptors, did not significantly affect locomotor activity. The pronounced locomotor depression elicited by intra-ACB injections of CGS 21680 were completely blocked by I.P. pretreatment with DMPX, an adenosine receptor antagonist exhibiting selectivity for striatal A2 receptors, at a dosage which alone had no significant effect on locomotor activity. Adenosine A2a receptors in the nucleus accumbens may selectively modulate dopamine-mediated mesolimbic behavioral circuits involved in spontaneous locomotion.

Adenosine A2a receptors in the nucleus accumbens mediate locomotor depression.

The effects on locomotor activity (LA) of selective agonists for adenosine receptor subtypes were examined in mice following bilateral injections into the nucleus accumbens (ACB). The ACB is not only richly innervated by dopaminergic (DA) terminals but also exhibits the highest densities of adenosine A2a receptors in the brain. CGS 21680 (2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosi ne), a potent and highly selective adenosine A2a receptor agonist, elicited pronounced, dose-related reductions in LA (ID50 dosage = 0.0031 nmol/mouse). NECA (5'-N-ethylcarboxamidoadenosine), a mixed adenosine receptor agonist which exhibits high selectivity and potency at striatal A2a receptors, similarly elicited dose-related reductions in LA (ID50 dosage = 0.0023 nmol/mouse). In contrast, intra-ACB injections of CPA (N6-cyclopentyladenosine), a highly selective agonist for adenosine A1 receptors, did not exert any significant effects on LA, even at 2.0 nmol/mouse, a dosage at which both CGS 21680 and NECA depressed LA by almost 90% compared to vehicle controls. Further, the pronounced locomotor depression elicited by intra-ACB injections of both CGS 21680 and NECA, at approximately the ID65 dosage, was significantly antagonized by IP pretreatment with DMPX, (3,7-dimethyl-1-propargylxanthine), an adenosine receptor antagonist with selectivity for A2 receptors in the striatum, at a dosage (0.15 micromol/mouse) [corrected] which alone had no significant effect on LA. These observations provide support for the notion that adenosine may selectively modulate DA-mediated mesolimbic behavioral circuits via agonist actions at a population of A2a receptors densely concentrated in the ventral striatum.

Effects of combined acetylcholinesterase inhibition and serotonergic receptor blockade on age-associated memory impairments in rats.

We recently reported that post-training administration of serotonergic receptor antagonists attenuated the inhibitory-avoidance memory deficits normally exhibited by aged rats. In the present study, we determined whether a subeffective dose of the serotonergic type-2 receptor antagonist, ketanserin, would augment the facilitative effects produced by the acetylcholinesterase inhibitor, physostigmine, on memory in aged rats using the same task. The drugs were injected intraperitoneally alone, or in combination, immediately following training. Retention testing occurred 24 hours following training. A dose-dependent enhancement of memory was demonstrated as a result of the two treatment conditions (physostigmine 0.01-10.0 micrograms/kg, ketanserin 1.0 mg/kg + physostigmine 0.001-0.01 micrograms/kg). The facilitation of memory produced by the combined treatment was observed at doses well below those required to produce a similar effect when each drug was administered alone. The results provide additional evidence for an interaction between the cholinergic and serotonergic neurotransmitter systems in learning and memory, and may have important implications in the treatment of age-related memory impairments.

N6-cyclopentyladenosine impairs passive avoidance retention by selective action at A1 receptors.

The effects of N6-cyclopentyladenosine (CPA), a highly selective agonist for adenosine A1 receptors, on retention of one-trial inhibitory avoidance behavior were examined in mice. Water-deprived animals were trained to avoid drinking by pairing foot-shock with licks from a water spout. Retention was measured as the suppression of drinking (latency to drink) 48 h following training. Administration of CPA (0.15-2.25 mumol/kg) 30 min prior to training produced a dose-dependent impairment in memory of the original avoidance task. The CPA-elicited deficits in retention performance were blocked by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A1 receptor antagonist; DPCPX (15 mumol/kg) administration alone had no effect on retention performance. These findings suggest that selective activation of a presumably central population of A1 receptors may impair retention performance and influence information processing.

Effects of combined serotonin depletion and lesions of the nucleus basalis magnocellularis on acquisition of a complex spatial discrimination task in the rat.

The purpose of the present experiment was to determine the effects of lesions of cholinergic neurons originating from the nucleus basalis magnocellularis (NBM), alone or in combination with central serotonin depletion, on learning and memory in rats trained in the Stone 14-unit T-maze--a complex, positively-reinforced spatial discrimination task. Lesion of cholinergic neurons within the NBM was accomplished by bilateral infusion of ibotenic acid. Serotonin depletion was accomplished by the systemic administration of p-chloroamphetamine (PCA). The results show that PCA-induced serotonin depletion enhanced learning. This effect was completely prevented by NBM lesions, despite the fact that NBM lesions alone did not affect the performance of rats in this task. The results of this study support the view that the cholinergic and serotonergic systems may functionally interact in learning and memory processes. The significance of this interaction in the etiology and treatment of dementia should be further investigated.

Enhanced spatial discrimination learning in rats following 5,7-DHT-induced serotonergic deafferentation of the hippocampus.

Learning in rats trained in the Stone 14-unit T-maze, a complex, positively reinforced spatial discrimination task was assessed following cytotoxic (5,7-dihydroxytryptamine; 5,7-DHT) deafferentation of the serotonergic inputs to the hippocampus. Serotonergic deafferentation was accomplished by infusing the cytotoxin in to the fornix-fimbria/cingulum bundle. Lesioned rats reached criterion (i.e. learned) in significantly fewer trials and made significantly fewer errors throughout training than either vehicle-injected or sham-operated controls. This represents the first time that the effects of selective chronic destruction of serotonergic inputs to the hippocampus have been investigated. The present results provide, therefore, evidence in support of a neuromodulatory role for serotonin (5-HT) within the rat hippocampus in the mediation of the processes underlying learning and memory for this task. Other studies are, therefore, warranted in order to determine whether hippocampal 5-HT also plays a role in the mediation of the processes underlying learning and memory in other types of tasks.

The effects of p-chloroamphetamine, a depletor of brain serotonin, on the performance of rats in two types of positively reinforced complex spatial discrimination tasks.

Learning in male Sprague-Dawley rats was assessed in two types of positively reinforced complex spatial discrimination tasks (Stone 14-unit T-maze and eight-arm radial-arm maze) following cytotoxic lesions of central serotonergic terminal projection fields with p-chloroamphetamine (PCA). Learning, as expressed as mean number of errors per day and mean number of trails required to reach criterion, was significantly enhanced in the PCA-lesioned animals trained in the Stone maze. On the other hand, the performance of the PCA-lesioned animals trained in the eight-arm radial-arm maze was not found to differ significantly from that of saline-injected animals. The improved acquisition of the PCA-lesioned rats trained in the Stone maze was completely abolished following pretreatment with the selective serotonergic reuptake inhibitor norzimeldine. Neurochemical analyses of the brains of representative animals revealed that the levels of serotonin and its major metabolite, 5-hydroxy-3-indoleacetic acid, were both significantly reduced by PCA in all regions examined. While it is clear from these and other studies that the serotonergic nervous system plays an important role in the processes underlying learning and memory, these results further underscore the selective role of this neurotransmitter system in the way information is processed by the brain.

Potentiation of oxotremorine-induced hypothermia by alaproclate in PCA lesioned and non-lesioned rats.

Stimulation of muscarinic cholinergic receptors with the highly potent and selective receptor agonist oxotremorine produced hypothermia in rats. Alaproclate, a purported selective serotonergic reuptake inhibitor, potentiated this response. Destruction of central presynaptic serotonergic terminals with the potent cytotoxin p-chloroamphetamine (PCA) failed to attenuate the hypothermic response to oxotremorine in alaproclate-pretreated animals. These results could be taken to suggest that alaproclate may act, at least in part, via a non-serotonergic mechanism to potentiate the oxotremorine-induced hypothermic response.

What is the nature of the role of the serotonergic nervous system in learning and memory: prospects for development of an effective treatment strategy for senile dementia.

The serotonergic nervous system has long been suspected of playing an important role in the processes underlying learning and memory. However, owing frequently to inconsistent and divergent results, its precise role is difficult to define. Recently, there has been a renewed interest in this neurotransmitter's role in the processes underlying learning and memory. This has, in part, been due to the observations that this system appears to undergo significant deterioration as a result of the pathology associated with certain age-related cognitive disorders as well as the discovery of multiple receptor subtypes. Reviewed here are the results of a number of studies designed to gain further insight into the role the serotonergic nervous system plays in learning and memory. A variety of methods are used to manipulate this system, and the effects of these manipulations on performance in a variety of behavioral tasks are summarized. Consistent with past observations, it is difficult to incorporate the results of the present series of studies into a single unified theory of serotonin's role in learning and memory. However, it is clear that not all of the inconsistencies can be attributed to differences in the methods used to manipulate the system or in the types of tasks used to assess learning and memory. Some of the inconsistencies clearly belie a selective role of this neurotransmitter in the processes underlying learning and memory and further underscore the complex nature of this system's role in the processing of information by the brain.

Enhanced passive avoidance retention following posttrain serotonergic receptor antagonist administration in middle-aged and aged rats.

The experiments examined the ability of posttrain administration of serotonergic receptor antagonists to attenuate the age-related deficits in avoidance retention normally exhibited by middle-aged (12 months) and aged (22 months) rats. Ketanserin (0, 1.0, 10.0 mg/kg) produced a significant dose-dependent increase in test step-through latencies in both age groups. The suppression of responding did not appear to be due to generalized learned aversion as nonshocked rats, injected with the highest dose of ketanserin, did not exhibit similar elevations in test latencies. In order to determine whether the ketanserin-induced effect could be generalized to other serotonergic antagonists, middle-aged and aged animals were injected with a single dose of mianserin (10.0 mg/kg). This antagonist also significantly increased step-through latencies in both age groups, while not affecting the performance of nonshocked animals. The results provide additional evidence for a role of the serotonergic nervous system in memory, and may have important implications in the development of effective treatment strategies for geriatric-related cognitive disorders.

Different temporal effects of serotonergic antagonists on passive avoidance retention.

The experiments examined the effects of acute administration of three different serotonergic receptor antagonists (ketanserin, pirenperone and mianserin) on one-trial passive avoidance retention in mice. Administration of each antagonist 30 min before training produced a dose-dependent impairment in retention. In contrast, administration of each of the antagonists immediately after training produced a dose-dependent improvement in retention. The time-dependent effects of pre- and post-train antagonist administration were assessed using pirenperone. In both cases, the effects on test performance were determined to be time-dependent. The results provide additional evidence suggestive of a differential role of the serotonergic nervous system in the processes underlying learning and memory.

Evidence for a possible interaction between noradrenergic and serotonergic neurotransmission in the retrieval of a previously learned aversive habit in mice.

The present series of experiments examined the effects of pretreating pirenperone-injected mice with a variety of non-serotonergic receptor antagonists on retrieval of a one-trial inhibitory (passive) avoidance task. Water-deprived mice were trained to avoid drinking from a water spout located in the avoidance chamber by pairing foot-shock with licks from the water spout. Retention was measured as the suppression of drinking (latency to drink) 48 h later. Pre-test administration of pirenperone (1.0 mg/kg) significantly enhanced retrieval (increased latencies). The suppression of drinking could not be attributed to the non-specific effects of pirenperone on behavior in general, as the performance of non-contigently shocked mice injected with the same dose of pirenperone did not exhibit a similar elevation in latencies. Of the seven pretreatment drugs examined, only phenoxybenzamine (1.0 mg/kg) completely blocked the pirenperone-induced response. Bicuculline (1.0 mg/kg) partially attenuated the enhanced performance resulting from pre-test pirenperone administration. The results suggest that the pirenperone-induced response may be partly due to activation of noradrenergic (alpha) neurotransmission.

Enhancement of the memory of a previously learned aversive habit following pre-test administration of a variety of serotonergic antagonists in mice.

The present study examined the effects of pre-test administration of a number of serotonergic receptor antagonists on the retrieval of a previously learned aversive habit in the mouse. All of the receptor antagonists (pirenperone, ketanserin, mianserin, methysergide and metergoline) produced a dose-dependent increase in the latency to complete 5 s of drinking 48 h after training. This suppression of drinking could not be attributed to nonspecific effects of the drugs on behavior (e.g., illness, reduced thirst, or activity), as non-contingently trained mice failed to exhibit similar elevations in their test scores. These results are, therefore, further support for an important role for serotonin in the processes underlying learning and memory.

Evidence for angiotensin II receptors in the urinary bladder of the rabbit.

Dose-response (DR) curves for several angiotensin analogs were examined on isolated rabbit detrusor strips with washout and rest between each addition. The order of potency was [Val5]-angiotensin II greater than [Ile5]-angiotensin II greater than [Ile5]-angiotensin I greater than [Val4]-angiotensin III. Repeated cumulative DR to [Val5]-AII resulted in a gradual increase in potency and intrinsic activity for four DR. However, the maximum force generated occurred at lower agonist concentrations and was less than that of the single methods, suggesting tachyphylaxis. Atropine (1.0 microM) shifted the cumulative DR curve downward, suggesting some cholinergic component possibly involving a presynaptic site of action. The magnitude of field-stimulated atropine-resistant contractions was reduced by both 1.0 and 10 microM saralasin as well as 10 microM naloxone. Tissue binding with 125I-labelled angiotensin II on isolated detrusor smooth muscle membranes indicated specific binding saturation occurred at 14.3 fmol/mg with a KD of 0.72 nM in EDTA-Tris buffered saline. Thus our results show that angiotensin II (AII) receptors can be demonstrated in destrusor muscle by ligand binding experiments on cell membranes and that saralasin and naloxone partially block atropine-resistant contractions. However, it seems unlikely that AII serves as a neurotransmitter because of the delay in onset of action of exogenous AII in isolated bath experiments and the apparent inability of saralasin to totally abolish the atropine-resistant field-stimulated preparation. If AII serves a role in neurotransmission it most probably is as a neuromodulator.

Chronic oesophageal fistulation for studying oropharyngeal and gastric influences on ingestive behaviour in the pigeon.

Surgical and postoperative procedures were developed in the pigeon to study the influence of oropharyngeal and postingestional factors in the control of food and water intake. Pigeons were prepared with an oesophageal fistula so that material swallowed emerged from a tube exteriorized at the neck. Nutrient solutions were injected into the lower portion of the oesophagus through a feeding tube inserted in the fistula. A postoperative tube-feeding regimen, using commercially available laboratory chow, was established to maintain the pigeon at its preoperative weight.

Chronic intracarotid cannulation of pigeons for administration of behaviorally active peptides.

Chronic intracarotid cannulation of the common carotid artery was performed in the pigeon. The catheter system of polyethylene tubing consisted of an indwelling component and an injection component. The indwelling component was exteriorized at the occiput so the bird could not reach the catheter with its beak. Following surgery, the pigeons were housed individually and received food and water ad libitum and no special care was necessary. The catheter was flushed daily with heparin in 0.9% NaCl solution to maintain patency. 30 pigeons were continuously or intermittently infused with bioactive peptides for up to 60 days after cannulation.