Evidence for adrenergic neurons in a memory access pathway

Injection of a beta-adrenergic blocker, propranolol, in rats within 5 min after training of a step-down passive avoidance response had no effect on performance either 2 or 6 hr later, however, when testing occurred 1, 3 or 7 days after training and injection a significant performance decrement was observed. If drug injection was postponed until 1 or 3 days after training and testing was conducted 2 hr later, again poor avoidance performance was obtained. No support for a state-dependency explanation [14] of the propranolol amnesia could be found. The amnesia that followed beta-adrenergic receptor block was identical to that previously reported when norepinephrine biosynthesis was reduced [9] and supports the hypothesis of a role for adrenergic neurons in memory formation and retrieval that is different from cholinergic neurons [4,10].     

DDC-Induced amnesia and norepinephrine: A correlated behavioral-biochemical analysis

Diethyldithiocarbamic acid (DDC), a dopamine-B-hydroxylase inhibitor, when injected into rats 30 min to 6 h before training of a passive avoidance task, impaired formation of long-term memory as indicated by performance on a retention test 24 h later. Performance of the task was at its minimum when injection occurred 2 to 4 h prior to training; recovery was evident in animals trained 5 or 6 h after drug treatment. Catecholamine assay of brains of temporally yoked animals showed that norepinephrine depletion followed a time course paralleling that of the amnesia. These findings support the hypothesis that the degree of memory storage, as reflected in performance following training in a passive avoidance task, can be directly correlated with the level of norepinephrine existing at the time of training.     

DDC-induced retrograde amnesias prevented by injections of dl-DOPS.

Injection of a dopamine beta-hydroxylate inhibitor, diethyldithiocarbamate (DDC) in rats 30 min prior to training of a step-down passive avoidance task impaired performance of the task 24 hr later. Similarly, injection of DDC 30 min prior to testing blocked retrieval of a passive avoidance habit trained in normal rats the previous day. Injection of a direct norepinephrine (NE) precursor, dl-thero 3,4-dihydroxyphenylserine (DOPS) 60 min before DDC prevented both amnesias. These data support the hypothesis that reduced levels of NE are responsible for DDC-induced amnesias.     

Locus coeruleus and labile memory

Memory lability is defined as the period of time recently-formed memory remains susceptible to experimental modification. Electrolytic lesions delivered through chronic indwelling electrodes to the locus coeruleus (LC) complex of mice, made shortly after learning, resulted in an extension of memory lability. Mice with unilateral, but not bilateral LC damage became amnesic following electroconvulsive shock (ECS) administered 7 days (168 hr) after memory formation. ECS had no effect on memory in LC-lesioned mice when administered 14 days following training. In a second experiment, the temporal relationship between time of memory formation and time of LC damage was found to be critical to the occurrence of this extended period of lability. In a third experiment, we tested the possibility that prolonged trace lability was the result of weaker memory formation as reflected by decreased persistence (i.e. faster forgetting) of the memory. The results indicated equal rates of forgetting for normal and LC-lesioned mice. Present results support the hypothesis that the locus coeruleus complex normally plays an important role in delimiting the time-course of initially labile stages of memory. By inference, these data suggest further that such a delimiting function of the locus coeruleus is mediated through its noradrenergic modulation of other brain regions.     

Effects on retention of posttraining amphetamine injections in mice: Interaction with pretraining experience

These experiments examined the effects of d-amphetamine on retention of one-trial inhibitory (passive) avoidance training in mice. Water-deprived mice were pretrained to lick from a water spout at the end of a darkened compartment. Footschock was administered during licking after 4, 6, or 7 days of pretraining. Retention performance (latency to lick) was measured 24 h after training. The effects on memory of posttraining amphetamine varied not only with amphetamine dose but also with the amount of pretraining. In animals pretraining for 7 days, 0.3 and 1.0 mg/kg but not 0.03, 0.1, or 3.0 mg/kg posttraining amphetamine significantly enhanced later retention performance. In mice pretrained for 6 days, 1 mg/kg amphetamine also enhanced retention performance. However, in mice pretrained for only 4 days, 1 mg/kg amphetamine impaired later retention performance. These results are consistent with the view that posttraining treatments may affect memory storage processes by interacting with training-related arousal levels.     

Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures

The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine β-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout (Dbh −/−) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced seizures (CIS) by inhibiting DBH. To test this, we examined CIS in wild-type and Dbh −/− mice following administration of disulfiram or the selective DBH inhibitor nepicastat. We found that Dbh genotype had no effect on CIS probability or frequency, whereas disulfiram, but not nepicastat, increased the probability of having CIS in both wild-type and Dbh −/− mice. Both disulfiram and nepicastat increased CIS frequency in wild-type but not Dbh −/− mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated Dbh −/− mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two distinct mechanisms: it both increases CIS frequency by inhibiting DBH and increases CIS frequency in a DBH-independent manner.     

Neurochemical mediation of reward: A significant role for dopamine?

Intraventricular injection of 6-hydroxydopamine resulted in only a temporary decrement of self-stimulation. Treatment with the alpha-adrenergic receptor blocking agent, phentolamine, had virtually no effect on self-stimulation in either 6-hydroxydopamine treated or normal rats; whereas haloperidol, a dopamine receptor blocking agent, markedly reduced self-stimulation in normal rats. Treatment with FLA-63, a potent inhibitor of dopamine-beta-hydroxylase, had no effect on self-stimulation but very significantly reduced eating. These results imply that dopamine may be importantly involved in the mediation of positive reinforcement and/or that the role of norepinephrine may be a minor one.     

Memory retention: effect of prolonged cholinergic stimulation in mice

The fundamental hypothesis that drugs may affect memory processing by prolonging transmitter action was tested by extending the time of drug action, using repeated administrations of the cholinergic agonist, arecoline hydrobromide (ARE). The ARE was injected intracerebroventricularly into mice immediately after training (T-maze footshock avoidance) and at 90-min intervals thereafter, for a total of 1, 2, or 3 injections. The results indicate that 1 injection had no effect whereas 3 successive injections significantly improved memory retention test performance. The results confirm the hypothesis being tested; six control groups ruled out other plausible interpretations of the results.     

Tyramine in the assessment of regional adrenergic function

Regional adrenergic function is difficult to assess in humans. Tyramine given through a microdialysis probe may be a useful tool in this regard. However, tyramine data is hard to interpret given the drug's complex mode of action. We characterized the response to tyramine, isoproterenol, and dopamine in adipose tissue with microdialysis probes in normal subjects. We measured glycerol concentrations to follow changes in lipolysis and monitored tissue perfusion with ethanol dilution. During perfusion with tyramine, dialysate glycerol concentration increased dose-dependently from 83+/-8 microM at baseline to 181+/-18 microM at 3.5 mM tyramine (p<0.001) followed by a fall down to 121+/-9 microM at 35 mM tyramine (p<0.001). Propranolol almost completely blocked this response. A similar lipolytic response was not observed in isolated human adipocytes. Dopamine <35 microM did not replicate the tyramine-induced lipolysis; however, dopamine >35 microM potently inhibited lipolysis. We conclude that tyramine-induced lipolysis is explained by a pre-synaptic mechanism. Tyramine applied through a microdialysis probe in concentrations up to 3.5 mM can be used to assess pre- and post-synaptic mechanisms regulating lipid mobilization.     

Effect of cycloheximide and d-amphetamine on brain catecholamines in two mouse strains

The ability of cycloheximide to inhibit brain catecholamine synthesis in C57BL/6J and DBA/2J mice was studied to determine whether differences exist in these two strains with regard to this action and whether such effects correlate with previously reported differences in sensitivity to the amnesic effects. Administration of cycloheximide caused a dose-dependent inhibition of norepinephrine and dopamine synthesis in both strains. There was a significant effect due to strain on dopamine synthesis in drug-treated animals. d-Amphetamine partially prevented the decrease in the rate of synthesis of norepinephrine, dopamine and normetanephrine caused by cycloheximide in the C57 strain but enhanced the inhibition of synthesis of these compounds in the DBA strain. The results suggest that the reported differences in sensitivity to the behavioral effects of cycloheximide may be associated with the degree of inhibition of catecholamine synthesis in these two mouse strains.     

Memory effect of dl-threo-3,4-dihydroxyphenylserine (DOPS) in human Korsakoff's disease

A group of amnesic patients with Korsakoff's disease were treated with a single 1 g dose of dl-threo-3,4-dihydroxyphenylserine (DOPS) and placebo (lactose) in a double-blind crossover study. Three hours following administration, patients were given a battery of psychometric tests to determine the effects of the treatment on memory functions. Administration of DOPS had a significant effect on performance on the Memory Passages test but not on any of the other measures of memory. The effect of DOPS on Memory Passages is similar to the response observed following administration of clonidine in Korsakoff patients. Blood pressure and pulse, measured before and every 2 h after treatment, were unaffected by DOPS.     

An autoradiographic method of mapping the distribution and density of monoamine neurons in mouse brain.

A combined in vitro uptake and autoradiographic procedure as an important complement to the histochemical fluorescence method is described. Slabs of fresh mouse brain were incubated with 14C-NE, 14C-DA or 14C-5-HT, freeze-dried, and placed against X-ray film for autoradiography, Catecholamine nerve terminals were labeled by in vitro incubation with 14C-NE or 14C-DA. Dopaminergic terminals were labeled by 14C-NE incubation preceded by desipramine (to block uptake into NE terminals). With 14C-5-HT incubation, the uptake pattern indicated the possibility that 5-HT nerve terminals were being labeled. Advantages of this method are that it allows the visualization of overall density and distribution of selected monoamine nerve terminals or uptake sites of other putative neurotransmitters in whole coronal or sagittal sections, so that data are obtained from many areas of brain or spinal cord rather than in only those areas preselected for microscopic viewing.     

Behavioral effects of intracerebrally administered catecholamines in mice and their modifications by some adrenergic blocking agents

Increasing doses of epinephrine (E), norepinephrine (NE) and dopamine (DA) were administered intracerebrally to mice.These drugs cause, at low doses (1–10 g/mouse) purely excitatory symptoms, while at high doses (30–100 g/mouse) they induce a biphasic syndrome, consisting of an initial period of excitation, followed by depression. In animals pretreated with -adrenolytics (yohimbine and phentolamine) the excitation caused by the low doses of the catecholamines was unaffected, while the running fits and the depression elicited by the high doses were attenuated or abolished.     

The role of neurotransmitters in stress-induced antinociception (SIA)

The role of the neurotransmitters, norepinephrine, dopamine and serotonin in stress-induced antinociception (SIA) was examined by altering neurochemical tone with appropriate pharmacological tools. Quipazine (15.0 mg/kg, IP) a serotonin agonist, increased the peak and duration of antinociception following stress and BC-105 (3.0 mg/kg, IP), a serotonin antagonist, blocked the increase of tail-flick latency following stress. Clonidine (0.1 mg/kg, SC) an alpha 2 agonist, markedly decreased SIA whereas phenoxybenzamine (2.5 mg/kg, IP), an alpha 1 antagonist, increased the peak and duration of SIA. When dopaminergic tone was increased with apomorphine (0.05 mg/kg, SC) the increase of tail-flick latency after stress was markedly attenuated whereas blockage of dopamine receptors with haloperidol (2.5 mg/kg, IP) increased the peak and duration of SIA. Alterations of serotonergic, but not noradrenergic or dopaminergic, tone had similar effects on increased latency in tail-flick test produced by brain stimulation produced analgesia (SPA), morphine and SIA. These data support the hypothesis that alterations in tail-flick latency involves a serotonergic system.     

Possible role of dopamine-beta-hydroxylase in the regulation of norepinephrine biosynthesis in rat brain.

In Experiment 1, the dose-response effects of three dopamine-beta-hydroxylase (DBH) inhibitors (diethyldithiocarbamate, FLA-63 and U-14, 624) on the endogenous levels of norepinephrine and dopamine in pons-medulla of rat brain were determined. In Experiment 2, the effect of low doses of diethylithiocarbamate (2.5 to 120 mg/kg) on the level of norepinephrine-3H produced from dopamine-H3 was determined. The data obtained by extrapolation of the curves in both experiments provided an estimation of the in vivo level of DBH activity and suggested that it was not present in excess. Finally, in Experiment 3, the three DBH inhibitors reduced self-stimulation (a behavior dependent upon catecholamines) in a dose-related manner and intraventricular injections of 1-norepinephrine reinstated normal rates of self-stimulation. The results from the three experiments are consistent with the idea that DBH is involved in the regulation of norepinephrine biosynthesis. The relationship of this finding to our earlier report of a deficit of DBH in post-mortem brains of schizophrenics is discussed.     

Memory facilitation with posttrial injection of oxotremorine and physostigmine in mice

The immediate posttrial injection of oxotremorine (0.125, 0.250 and 0.500 Mol/kg i.p.) and equimolecular doses of physostigmine can facilitate the retention of a passive avoidance response in mice. Injections given 10 min after training also significantly facilitate retention, but injections given 30 or 120 min after training do not affect retention. These findings suggest an action of oxotremorine and physostigmine on mechanisms involved in memory storage. The enhanced retention produced by oxotremorine and physostigmine was blocked by pretreatment with atropine (2 Mol/kg, 20 min, i.p.) but was not affected by methylatropine (2 Mol/kg, 20min, i.p.). The retention was not modified by posttrial injection of metoxotremorine (0.250 Mol/kg i.p.) or neostigmine (0.250 Mol/kg i.p., quaternary analogs of oxotremorine and physostigmine, respectively. The results suggest a central action of both cholinergic agents attributable to an activation of muscarinic brain receptors.     

6-Hydroxydopamine and avoidance: Possible role of response suppression.

The intraventricular administration of 6-HD to rats pretreated with pargyline resulted in severe, long-lasting decreases in avoidance responding with little or no effect on escape responding. Despite the fact that the rats failed to avoid, they appeared to be able to discriminate the CS, as was evident from freezing behavior and other symptoms of an apparent fear reaction during the CS. The increase in freezing, a response that was incompatible with avoidance was seen during the first few test sessions after 6-HD treatment and seemed to be largely responsible for a gradual decline in avoidance responding during this same period. The role of CA depletion in the animal's response to aversive stimuli thus appears to be a significant aspect of the avoidance decrement that follows 6-HD administration.     

Amnestic potency of proline analogs correlates with anti-spreading depression potency

L-Proline and some of its analogs have been shown to prevent spreading depression (SD) in the chick retina at relatively low concentrations and to impair memory processing without provoking toxic or electrophysiological disturbances. Both effects are hypothesized to be caused by inhibition of the effects of glutamate released into the extracellular space. L-Proline, its D-enantiomer, six proline analogs including two homologs (L-azetidine-2-carboxylic acid and DL-pipecolic acid), and five other compounds were examined for their effects on spreading depression and their amnestic and electrophysiological effects. L-Proline, L-baikiain, DL-3,4-dehydroproline, and L-4-hydroxyproline all reduced the incidence of SD in the chick retina and proved to be amnestic. D-Proline, L-pyroglutamic acid, L-azetidine-2-carboxylic acid, DL-pipecolic acid, L-glutamic acid diethylester, L-isoleucine and L-norleucine neither depressed SD nor caused retrograde amnesia. L-Prolyl-L-proline and L-glutamine did not depress SD at low concentrations but had significant amnestic effects. None of the listed compounds induced EEG disturbances. Implications for memory mechanisms are discussed in the light of these results.     

Memory enhancement: Supra-additive effect of subcutaneous cholinergic drug combinations in mice

The amnesias characteristic of Alzheimer's disease and other age-related dementias are refractory to conventional pharmacotherapy. a recent treatment strategy is to combine present drugs to improve their memory enhancing effect. We used mice weakly trained on active avoidance in a T-maze to compare the effect of cholinergic drugs, given alone and in two-drug combinations, on retention test performance. All drugs were injected SC immediately after training. Memory retention was tested 1 week later. A dose-response curve was determined for each of four drugs (arecoline, edrophonium, oxotremorine, tacrine) and for each of the six possible two-drug combinations. Each drug and each combination improved retention test performance up to an optimal dose; the improvement decreased with further increases in dose. A striking reduction (66.2%–95.7%) in the optimal dose for enhanced retention was observed with these two-drug combinations.     

Comparative study of retrograde amnesia in rats on active and passive avoidance tasks and spontaneous recovery of memory

Naive and pretrained rats were trained in two active avoidance paradigms using a pole-climbing box and in a single-trial passive avoidance task using a T-maze. They were then subjected to amnestic treatments with electroshock, leptazol, pentobarbitone, or ether anesthesia. Single retention tests were given at 20–24, 44–48, or 68–96 h postreatment. Electroshock and leptazol seizures produced retrograde amnesia in all three paradigms, provided that seizures were maximal and retention was tested before 48 h. Prior treatment with anticonvulsant drugs prevented amnesia. Ether and pentobarbitone anesthesia failed to produce amnesia in all three tasks. A trend of recovery from amnesia was observed in the electroshock and leptazol groups when tested for retention 48–96 h posttreatment. On the other hand, the nonamnesic control, pentobarbitone, and ether groups showed signs of forgetting at these longer intervals. Consolidation failure and/or retrieval block was surmised to be the cause of amnesia; recovery was the possible result of removing the block.