Administration Of Protein Synthesis Inhibitor Research Articles

Protein synthesis inhibitor administration before a reminder caused recovery from amnesia induced by memory reconsolidation impairment with NMDA glutamate receptor antagonist

Memory recovery in amnestic animals is one of the most poorly studied processes. In this paper, we examine the role of protein synthesis and a reminder in the mechanisms of amnesia and memory recovery in grape snails trained to conditioned food aversion. Amnesia was induced by the impairment of memory reconsolidation using NMDA (N-methyl d-aspartate) glutamate receptor antagonists. In an early stage of amnesia (day 3), injections of protein synthesis inhibitors into animals combined with a reminder by a conditioned stimulus (CS) led to the recovery of aversive reactions to its presentation. Two types of changes in reactions to CS were revealed. In most animals, a persistent recovery of memory retrieval was found that lasted for at least 10 days. In other snails, aversive responses to CS persisted for 24 h. Isolated injections of inhibitors, injections of inhibitors and a reminder by the learning environment (without presenting a CS), usage of a differentiating stimulus instead of a CS, or inhibitor injections after the reminder did not affect the development of amnesia. The administration of protein synthesis inhibitors and a reminder in the late period after amnesia induction (10 days) did not affect its development or caused a short-term memory recovery. We suggest that amnesia is an active process that develops over time. The reminder induces the reactivation of the amnesia process dependent on protein synthesis, while the administration of protein synthesis inhibitors leads to the impairment of amnesia reactivation and recovery of the state formed before amnesia induction (i.e., recovery of conditioned food aversion memory).

Imbalance in cerebral protein homeostasis: Effects on memory consolidation

The long-standing hypothesis that memory consolidation is dependent upon de novo protein synthesis is based primarily on the amnestic effects of systemic administration of protein synthesis inhibitors (PSIs). Early experiments on mice showed that PSIs produced interference with memory consolidation that was dependent on the doses of PSIs, on the interval between drug injection and training, and, importantly, on the degree and duration of protein synthesis inhibition in the brain. Surprisingly, there is a conspicuous lack of information regarding the relationship between the duration of protein synthesis inhibition produced by PSIs and memory consolidation in the rat, one of the species most widely used to study memory processes. We found that, in the male rat, a single injection of cycloheximide, a commonly used PSI, produced a significant imbalance in protein homeostasis: an early inhibition of protein synthesis that lasted for at least one hour, followed by hyperproduction of proteins that lasted three days. We evaluated memory consolidation of inhibitory avoidance trained with either low or high intensity of foot-shock at the peaks of protein synthesis inhibition and protein hyperproduction. We found that, independent of the moment of training, the low-foot-shock groups showed amnesia, while the high-foot-shock groups displayed optimal memory performance. These results indicate that memory consolidation of relatively weak training is impaired by the inhibition or hyperproduction of protein synthesis, and that intense training overcomes this dysregulation of protein homeostasis allowing for memory formation probably through non-genomic mechanisms.

Administration of Protein Synthesis Inhibitor before Reminder Reverses Amnesia Induced by Memory Reconsolidation Impairment with 5-HT Receptors Antagonist.

Administration of 5-HT receptor antagonist to snails trained in conditioned food aversion prior to reminding of the conditioning stimulus caused amnesia. At the early period of amnesia (day 3), injections of protein synthesis inhibitor cycloheximide without reminder or reminder alone were ineffective. At the same time, injections of the inhibitor combined with reminder led to memory recovery; this effect in most animals persisted for at least 10 days. In the rest snails, aversive responses to presentations of the conditioning stimulus persisted for 2 days. Cycloheximide injection and reminder in 10 days after induction of amnesia did not affect its development or caused a transient memory recovery (2 days). We hypothesized that amnesia is an active process unfolding in time. One of mechanism of this process is reminder-induced and protein synthesis-depended reactivation of amnesia. Inhibitor of protein synthesis disturbed this reactivation and led to recovery of the initial memory of conditioned food aversion.

Protein synthesis inhibitors induce both memory impairment and its recovery

The involvement of protein synthesis in the mechanisms of conditioned food aversion memory impairment and recovery in grape snails was studied. It was found that protein synthesis inhibitor (cycloheximide) injections before a reminder by the conditioned stimulus (CS) caused amnesia development. Three days after amnesia induction, injections of cycloheximide or another protein synthesis inhibitor, anisomycin, combined with a reminder by four CSs resulted in memory retrieval, which was saved for 24 h. Cycloheximide injections and the administration of one CS as a reminder to an amnestic animals caused the memory expression only in response to this CS, while it was absent the next day. The isolated administration of a reminder or inhibitor injections without a reminder was not effective. It is suggested that amnesia is an active process and that one of its mechanisms may be a protein-dependent amnesia reactivation caused by a reminder. The administration of protein synthesis inhibitors led to impairment of amnesia reactivation and to recovery of the state formed before amnesia induction and thus to the recovery of conditioned food aversion memory.

A role for autophagy in long-term spatial memory formation in male rodents.

A hallmark of long-term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long-term memory formation without influencing short-term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long-term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc-51-like autophagy activating kinase-1 (Ulk-1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long-term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long-term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP-LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3-methyladenine (3-MA) or Spautin-1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT-Beclin-1) improved long-term memory. These results indicate that autophagy is not necessary for learning, but is required for long-term memory formation.

Protein synthesis is not required for acquisition, consolidation, and extinction of high foot-shock active avoidance training

Long-term memory of active avoidance in mice is not disturbed by administration of protein synthesis inhibitors (PSIs) when relatively high levels of training are used, whereas a detrimental effect is produced with lower levels of training. PSIs also disrupt extinction of avoidance behaviors in rodents, but it is not clear whether PSIs also affect this form of learning when the behavior to be extinguished was produced by a high level of training. Experiment 1 demonstrated that rats treated with the PSI cycloheximide (CXM) 30min before training developed normal acquisition after training with either high or low foot-shock stimulation, but that memory consolidation was hindered only after low foot-shock training. Experiment 2 demonstrated that CXM disrupted extinction when administered before the first of a series of extinction sessions when low foot-shock intensity was used during training; in contrast, after training with a higher foot-shock, the PSI treatment only interfered transiently with extinction. These results indicate that acquisition, consolidation, and extinction of active avoidance learning produced by high aversive stimulation are not dependent on protein synthesis and that these processes are governed by mechanisms different from those underlying moderate forms of learning.

Effects of Actinomycin D and Cycloheximide on Zinc Status in Marginally Zinc-Deficient Rats

During deficient zinc intake, rats are liable to suffer zinc deficiency under the following conditions: higher protein diet, diet containing higher quality (higher nutritive value) protein, and higher dietary intake. This suggests that a higher protein nutritional status (rapid increase in body protein) in rats leads to a lower zinc nutritional status (higher zinc requirement). In contrast, it is expected that a lower protein nutritional status (lowered body protein biosynthesis) is not liable to result in a lower zinc nutritional status. Therefore, the effects of protein biosynthesis inhibitors on zinc status were studied. Actinomycin D and cycloheximide were administered to rats under a marginally zinc-deficient condition. The growth of rats was depressed and serum and femur zinc concentrations were increased by administration of protein biosynthesis inhibitors. The carcasses of rats administered protein synthesis inhibitors had a higher zinc/protein ratio than those of the respective pair-fed (calorically equivalent to the zinc-deficient group) rats. Results suggest that zinc deficiency in rats is mainly alleviated by decreased food intake with administration of protein synthesis inhibitors. Furthermore, protein biosynthesis inhibition alone alleviated zinc deficiency.

Cycloheximide produces amnesia for extinction and reconsolidation in an appetitive odor discrimination task in rats

A considerable literature has shown deficits in memory resulting from the administration of protein synthesis inhibitors; however, most of the past literature in this field has focused on acquisition of new memory using aversively-motivated tasks. The effect of protein synthesis inhibition on appetitive learning and memory as well as extinction is less clear. The present study employed an appetitive odor discrimination paradigm to examine the effects of acute cycloheximide administration (1 mg/kg) on reconsolidation and extinction. Male, Long-Evans adult rats were trained to discriminate between two odors (i.e., cocoa and cinnamon) and then received extinction trials following an intraperitoneal injection of cycloheximide or vehicle. Twenty-four hours later, rats were tested via one non-reinforced test trial. Results showed amnesia for extinction as well as original training (i.e., correct odor choice) in cycloheximide-injected rats in this appetitive task, while vehicle-injected controls showed good memory for extinction. These data add to a growing literature showing the importance of protein synthesis inhibition for extinction and reconsolidation in appetitive learning and memory.

Enhanced inhibitory avoidance learning prevents the long-term memory-impairing effects of cycloheximide, a protein synthesis inhibitor

Interference with activity of numerous cerebral structures produces memory deficiencies; in many instances, however, when animals are over-trained such interference becomes innocuous. Systemic administration of protein synthesis inhibitors impairs long-term retention; this effect has been interpreted to mean that protein synthesis is required for memory consolidation, though little is known about the effect of protein synthesis inhibitors on memory of enhanced learning in the rat. To further analyze the protective effect of enhanced learning against amnesic treatments, groups of Wistar rats were trained in a one-trial step-through inhibitory avoidance task, using different intensities of foot-shock during training. Cycloheximide (CXM; 2.8 mg/kg), an inhibitor of protein synthesis, was injected either 30 min before training or immediately after training. Twenty-four hours after training retention latencies were recorded. Our data showed that both pre- and post-training administration of CXM produced amnesia in those groups that had been trained with relatively low foot-shock intensities, but no impairment in retention was observed when relatively high intensities of foot-shock were administered. These and similar results lead us to conclude that protein synthesis inhibitors may interfere with memory consolidation, but their effect disappears when animals are submitted to an enhanced learning experience, calling into question the idea that protein synthesis is required for memory consolidation.

Intravenous injection of cycloheximide induces apoptosis and up-regulates p53 and Fas receptor expression in the rat liver in vivo

A single administration of protein synthesis inhibitor, cycloheximide (CHX) induces apoptosis of hepatocytes in vivo. We investigated the underlying mechanisms of this phenomenon and the role of p53 and Fas receptor using terminal dUTP nick end labeling (TUNEL), quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Rat liver tissue specimens were obtained at different time intervals after injection of CHX. The proportion of TUNEL-positive apoptotic hepatocytes increased with time and reached a plateau at 2.5 h after the injection. The p53 and Fas receptor mRNAs and the proportion of immunoreactive p53-positive and Fas receptor-positive hepatocytes increased markedly with time from 1 h after the administration. Since the time course of increased proportion of apoptotic hepatocytes does not parallel that of p53- or Fas receptor-positive hepatocytes and apoptotic hepatocytes appeared prior to up-regulation of p53 and Fas receptor expression, it is likely that the enhanced expression of p53 and Fas receptor is not involved directly in CHX-induced apoptosis of hepatocytes in vivo. Rats injected with a single intravenous dose of CHX, however, provide a simple and useful model for investigating the apoptotic machinery and the molecular mechanism of transcriptional up-regulation of p53 and Fas receptor in hepatocytes.

Mechanisms of memory reorganization during retrieval of acquired behavioral experience in chicks: the effects of protein synthesis inhibition in the brain.

According to current concepts, memory can be disrupted by administration of protein synthesis inhibitors over a relatively short time period before and after learning. However, data have been obtained indicating that protein synthesis inhibitors can induce amnesia when given long after learning if administration is performed in reminder conditions, i.e., when the animal is presented with one of the environmental components which previously formed the learning situation. The aim of the present work was to confirm the possibility of inducing memory disruption in chicks at late post-learning stages by administering the protein synthesis inhibitor cycloheximide in association with a reminder procedure. Day-old chicks were trained to perform a standard passive avoidance task. Chicks were given cycloheximide (20 microg, intracerebrally) 5 min before the reminder procedure, which was performed 2, 24, or 48 h after training. Testing was conducted 0.5, 1, 3, 24, and 48 h after the reminder. Administration of cycloheximide in association with the reminder procedure induced the development of temporary amnesia, whose duration gradually decreased as the interval between training and reminding increased. These data led to the hypothesis that a memory reactivated by a reminder undergoes a process of reorganization and reconsolidation, which depends on the synthesis of new proteins. The quenching of the ability of protein synthesis inhibition during the reminder to disrupt memory demonstrates the existence of a gradual process resulting in consolidation of memory between 2 and 48 h of learning.

Activation of CPP-32 protease in hippocampal neurons following ischemia and epilepsy

Recent in vitro studies indicate an involvement of members of the interleukin-1β converting enzyme (ICE) family of proteases in programmed neuronal cell death. Cell death of hippocampal neurons in animal models of cerebral ischemia and epilepsy shows morphological features of apoptosis and can be prevented by administration of protein synthesis inhibitors suggesting that de novo synthesis of components of the cell death program is necessary for neuronal apoptosis. In the present study we demonstrate by in situ hybridization analysis that expression of CPP-32, an ICE-related protease, is significantly upregulated in CA1 hippocampal neurons following global ischemia induced by cardiac arrest and in hippocampal neurons of the CA3/CA4 region after kainate-mediated epilepsy, respectively. Moreover, an increase in CPP-32-like proteolytic activity was detected in hippocampal extracts 24 h after ischemia using the fluorogenic CPP-32 substrate Ac-DEVD-AMC. Activation of CPP-32 clearly preceded cell death of hippocampal neurons as assessed by in situ end-labelling of nuclear DNA fragments. These results indicate that CPP-32 protease may be activated at both the transcriptional and post-translational level during neuronal apoptosis and that activation correlates with the selective vulnerability of hippocampal pyramidal neurons to ischemic and epileptic insults.

Transitory effects of intracerebral administration of protein synthesis inhibitors on rat's spontaneous behavior.

In order to evaluate whether the effects of the local administration of protein synthesis inhibitors are transient or permanent, small volumes of cycloheximide, 2-deoxy-d-galactose, tetrodotoxin, and saline solution were stereotaxically administered in the amygdala, substantia nigra, and nucleus basalis magnocellularis of naive male adult Wistar rats. Three spontaneous activities (feeding, drinking, locomotion) were continuously recorded. 12 groups of animals were housed in activity cages for 6 consecutive days with a light/dark schedule (LD 12:12). The active compounds at doses sufficient to cause only limited effects were administered at the beginning of the light period of the fourth day. During the initial three days spontaneous activities remained constant, following the normal circadian distribution of rodents. Cycloheximide and tetrodotoxin administrations were followed by transient site-specific and activity-specific changes, which occurred only during the fourth and fifth day. The results show that protein synthesis inhibitors can be locally administered into the CNS at doses affecting only limited subcortical structures. The observed modifications of the three spontaneous activities were all transient and were always followed by complete recovery within 48 hours, thus confirming previous biochemical data. Therefore, the local injection of protein synthesis inhibitors, like tetrodotoxin, lidocaine or other compounds, can be employed to induce transient functional effects on well delimited brain structures in order to study the time-course of the mechanism of mnemonic trace formation.

Apoptotic cell death induced by optic nerve lesion in the neonatal rat

Cell death can be ascribed to one of two distinct modes of degeneration: apoptosis (programmed or active cell death) or necrosis (passive degeneration). While apoptosis is generally assumed to occur in physiological conditions such as normal development or tissue turnover, necrotic cell degeneration is induced in pathological situations. Here we report that also in a pathological situation, such as after axotomy in the CNS, apoptotic type of cell death comes into play: following intracranial transection of the optic nerve in the neonatal rat in vivo, retinal ganglion cells undergo an active, apoptotic cell death. In fact, the administration of protein synthesis inhibitors (actinomycin D and cycloheximide) prevents the appearance of pyknotic nuclei as well as of fragmented DNA of ganglion cells at 24 hr postlesion. Correspondingly, the number of surviving cells after actinomycin D and cycloheximide treatment is comparable to normal, unlesioned retinas. In addition, cycloheximide decreases the number of pyknotic ganglion cells during spontaneous cell death.

Sleep and medial reticular unit responses to protein synthesis inhibitors: Effects of chloramphenicol and thiamphenicol

This study utilized a newly developed combination push-pull cannula/microdrive-microwire device to record single-unit activity within the diffusion field of substances introduced into the brainstem. Single unit activity within the midbrain and pontine medial reticular formation (RF) and sleep were recorded following either perfusion or oral administration of protein synthesis inhibitors, chloramphenicol and thiamphenicol. Chloramphenicol administration led to significant reductions in the frequency, but not the duration, of individual REM episodes without altering slow-wave sleep. During control experiments in slow-wave sleep, incipient REM was characterized by substantial increases in medial RF unit activity which occurred in association with the appearance of PGO spikes. On the other hand, discharge rates of medial RF neurons were markedly attenuated by chloramphenicol but were not consistently affected by thiamphenicol. These findings suggest that reduced unit activity contributes to the REM-suppressive effects of protein synthesis inhibitors.

Effects of cycloheximide, a protein synthesis inhibitor, on mouse brain catecholamine biochemistry

Cycloheximide (CXM), a protein synthesis inhibitor, has been shown to result in a marked inhibition of central catecholamine (CA) synthetic mechanisms at doses that cause amnesia in animals. Unlike other inhibitors of CA synthesis no significant depletion of whole brain NE or DA concentrations was observed 0.75, 1, 2, 3, 4, 6, 17, or 24 hours after administration of CXM (120 mg/kg) to C57BL/6J mice. In order to investigate the underlying basis of maintenance of CA levels in face of CA synthesis inhibition, the effects of CXM on in vitro release of 3H-NE was studied in mouse hypothalamic slices. CXM, in a dose related manner, significantly inhibited the potassium stimulated release of NE from hypothalamic slices. Anisomycin, another protein synthesis inhibitor, similarly inhibited NE release. These studies further document the effects of protein synthesis inhibitors on CA mechanisms and suggest that disruption of CA biochemistry may play a role in the amnesia observed after administration of protein synthesis inhibitors.

Differences in multiple unit activity discharge rate during short and long REM sleep periods: effects of protein synthesis inhibition

Midbrain reticular formation multiple unit activity discharge frequency during short or long duration control REM periods was compared to that occurring following the administration of protein synthesis inhibitors. The results show that during control long REM sleep periods there is a sharp increase of unit frequency which begins a few seconds prior to onset of REM sleep. This increase is absent during control short REM periods and is blocked by anisomycin and chloramphenicol. Since these latter drugs affect the frequency of REM periods, it is suggested that they do so by inhibiting phasic activities such as bursts of multiple unit activity.

Modification of REM sleep and associated phasic activities by protein synthesis inhibitors

The sleep-wake cycle and multiple-unit activity following the administration of various doses of protein synthesis inhibitors was studied in unrestrained cats. Special care was taken to analyze the effects of those drugs on phasic and tonic rapid eye movement (REM) sleep periods. It was observed that protein synthesis inhibitors decreased specifically total REM sleep time, at the expense of wakefulness, without altering slow-wave sleep time. It was further noted that the reduction in REM sleep was the result of a decrease in the frequency of REM periods, rather than in the duration of each individual period. In addition, protein synthesis inhibitors decreased phasic REM sleep and notably prolonged tonic REM periods. Moreover the phasic bursts of multiple-unit activity normally seen during REM sleep in control recordings were practically absent during REM sleep after the administration of protein synthesis inhibitors. It is suggested that protein molecules may participate in the mechanisms which trigger REM sleep.

Effects of intratracheal installation of dactinomycin on pulmonary edema and phosphatase activity of the lung lavage fluid in rats

Intratracheal (i.t.) administration of protein synthesis inhibitors produced pulmunary edema. O course inhibitors studied, dactinomycin (act. D) was the most potent. Severity of lung damage due to act. D was dose- and almost age-related. Maximal intensity of pulmunary edema was reached on the 3rd day following administration and relained constant for 14 days. Histopathological studies revealed confluent edema of the entire lung. Pretreatment with act. D induced tolerance to an ID 100 edematogenic dose of thiourea. The effect of i.t. instillation of act. D appear to be localized in the pulmonary tissue. Lung lavage fluid collected fromn drug-treated rats had higher acid and alkaline phosphatase activities, higher protein content and more leukocyte infiltration than that of control.

Temporal Characteristics of Amnesia induced by Protein Synthesis Inhibitor: Determination by Shock Level

IT is widely accepted that cerebral protein synthesis is involved in “long term” but not “short term” memory. Evidence for this comes from experiments in which amnesia for a multiple-trial avoidance task is induced by administration of protein synthesis inhibitors before or shortly after training1–3. In mice4–6, the amnesia, which is permanent, does not appear until 3 h or more after training, leading to the notion of a “short term” memory, and is not produced if the criterion for learning is too severe. We have given mice a different learning task and used the protein synthesis inhibitor cycloheximide to induce amnesia that has such different characteristics as to warrant a re-evaluation of the hypothesis.