Chronic intermittent ethanol exposure alters CA1 synaptic transmission in rat hippocampal slices

We investigated the neuroadaptive changes in synaptic transmission in the CA1 region of the hippocampus as a result of chronic intermittent ethanol exposure. Male Wistar rats were exposed daily (14 h) to ethanol vapors (blood alcohol levels = 150-200 mg%) for 12-14 days, and synaptic field potentials elicited by Schaffer collateral stimulation were compared in hippocampal slices from control and chronic ethanol-treated rats. Excitatory postsynaptic responses of slices were recorded under three conditions: (i) normal physiological saline; (ii) continued presence of 33 mM (150 mg%) ethanol (chronic ethanol-treated rats only); (iii) acute exposure to 33 mM ethanol. When recorded in ethanol-free physiological saline, the mean amplitude of the dendritic synaptic potential and the somatic population spike were significantly smaller in slices from chronic ethanol-treated rats compared to slices from control rats. Under conditions of continuous ethanol exposure, somatic and dendritic synaptic responses of slices taken from chronic ethanol-treated rats were further depressed, suggesting that neural pathways in area CA1 remained sensitive to ethanol. Acute application of ethanol led to a more pronounced reduction of the mean somatic population spike amplitude in slices from chronic ethanol-treated rats than in slices from control rats. However, dendritic synaptic responses were unaffected by acute ethanol in slices from both control and chronic ethanol-treated rats. In addition, we examined the involvement of presynaptic mechanisms in the effects of chronic intermittent ethanol using paired-pulse protocols. When recorded in the continued presence of ethanol, slices from chronic ethanol-treated rats exhibited a significant reduction in paired-pulse facilitation of the dendritic synaptic response compared to slices from control rats, indicating a presynaptic component to the neuroadaptive effects of chronic intermittent ethanol exposure. Conversely, acute ethanol exposure resulted in an enhancement of paired-pulse facilitation of the dendritic synaptic response, an effect that was similar in slices from both control and chronic ethanol-treated rats. Paired-pulse facilitation of the somatic population spike amplitude was not altered by chronic ethanol treatment. However, acute ethanol exposure significantly enhanced paired-pulse facilitation of the somatic population spike in slices from chronic ethanol-treated rats. This effect of acute ethanol was not observed in slices from control rats. Paired-pulse inhibition was not significantly altered in slices from chronic ethanol-treated rats, suggesting that GABAergic inhibitory mechanisms were not involved in the neuroadaptive effects of chronic intermittent ethanol exposure. We suggest that chronic intermittent ethanol exposure can induce multiple neuroadaptive changes in synaptic transmission of CA1 pyramidal neurons that are detectable at both the pre- and postsynaptic levels. Alterations in paired-pulse facilitation indicate presynaptic changes involving the release of the excitatory neurotransmitter glutamate, whereas changes in dendritic synaptic responses suggest postsynaptic changes in the responsiveness of neurons to synaptic input. Moreover, differential effects of chronic ethanol treatment on synaptic responses recorded in the dendrites versus the somatic region implicate additional effects of ethanol on somatically located mechanisms of CA1 pyramidal neurons. Furthermore, we suggest that complete tolerance to ethanol does not occur in the CA1 region of the hippocampus following chronic intermittent ethanol exposure.     

Brief exposure to the G-protein activator NaF/AlCl3 induces prolonged enhancement of synaptic transmission in area CAl of rat hippocampal slices

Summary Rat hippocampal slices were exposed briefly (12–15 min) to AlF_4- (10 mmol/l NaF, 10 mol/l AlCl³). The effect on synaptic transmission in area CAl was measured using extracellular electrodes placed in the stratum pyramidale and stratum radiatum. During fluoride exposure, both spike and EPSP amplitude fell to very low levels. Upon washout, spike amplitude recovered beyond control values, and in half of the preparations a prolonged enhancement of spike amplitude (> 2 h) occurred. Similar modulation of EPSP slope indicated that these charges were primarily synpatic. If Al³⁺ was omitted from the F^--containing saline, enhancement of spike amplitude, when observed, was brief (20–30 min) and no enhancement of EPSP slope was seen. Omission of Ca²⁺ from the AlF_4--containing saline also abolished any long-lasting enhancement of synaptic transmission, though population spike amplitude in most slices showed a brief (20–30 min) stimulatory response. In preparations in which LTP had previously been saturated, synaptic transmission was not enhanced by exposure to AlF_4-. It is concluded that NaF/ACl₃ exposure induces an LTP-like process by G-protein activation, which involves recruitment of processes involved in LTP, possibly including an enhancement of Ca²⁺-influx.     

Early stress exposure impairs synaptic potentiation in the rat medial prefrontal cortex underlying contextual fear extinction

Traumatic events during early life may affect the neural systems associated with memory function, including extinction, and lead to altered sensitivity to stress later in life. We recently reported that changes in prefrontal synaptic efficacy in response to extinction trials did not occur in adult rats exposed to early postnatal stress (i.e. footshock [FS] stress during postnatal day 21–25 [3W-FS group]). However, identifying neurocircuitry and neural mechanisms responsible for extinction retrieval after extinction training have not been precisely determined. The present study explored whether synaptic transmission in the hippocampal-medial prefrontal cortex (mPFC) neural pathway is altered by extinction retrieval on the day after extinction trials using electrophysiological approaches combined with behavioral analysis. We also elucidated the effects of early postnatal stress on the synaptic response in this neural circuit underlying extinction retrieval. Evoked potential in the mPFC was enhanced following extinction retrieval, accompanied by reduced freezing behavior. This synaptic facilitation (i.e. a long-term potentiation [LTP]-like response) did not occur; rather synaptic inhibition was observed in the 3W-FS group, accompanied by sustained freezing. The behavioral deficit and synaptic inhibition observed in the 3W-FS group were time-dependently ameliorated by the partial N-methyl-d-aspartate (NMDA) receptor agonist d-cycloserine (15 mg/kg, i.p.). These findings suggest that the LTP-like response in the hippocampal-mPFC pathway is associated with extinction retrieval of context-dependent fear memory. Early postnatal stress appears to induce neurodevelopmental dysfunction of this neural circuit and lead to impaired fear extinction later in life. The present data indicate that psychotherapy accompanied by pharmacological interventions that accelerate and strengthen extinction, such as d-cycloserine treatment, may have therapeutic potential for the treatment of anxiety disorders, including posttraumatic stress disorder.     

Effects of ethanol on encoding, consolidation, and expression of extinction following contextual fear conditioning

Studies of contextual fear conditioning have found that ethanol administered prior to a conditioning session impairs the conditioned freezing response during a test session the next day. The present experiments examined the effects of ethanol on extinction, the loss of conditioned responding that occurs as the animal learns that a previously conditioned context no longer signals shock. Ethanol (1.5 g/kg) administered prior to single (Experiment 1) or multiple (Experiment 2) extinction sessions impaired extinction. Ethanol administered prior to a test session disrupted the expression of freezing after extinction (Experiments 3-5). There was some evidence that ethanol served as an internal stimulus signaling the operation of conditioning or extinction contingencies (Experiments 4-5). In Experiment 6, postsession injections of 1.5 g/kg ethanol had no effect on extinction with brief (3 min) or long (24 min) exposures to the context, but injections of 3 g/kg after long exposures impaired extinction. Together, these results indicate that ethanol affects extinction by acting on multiple learning and performance processes, including attention, memory encoding, and memory expression.     

Glycolysis prevents anoxia-induced synaptic transmission damage in rat hippocampal slices

Prolonged anoxia can cause permanent damage to synaptic transmission in the mammalian CNS. We tested the hypothesis that lack of glucose is the major cause of irreversible anoxic transmission damage, and that anoxic synaptic transmission damage could be prevented by glycolysis in rat hippocampal slices. The evoked population spike (PS) was extracellularly recorded in the CA1 pyramidal cell layer after stimulation of the Schaffer collaterals. When the slice was superfused with artificial cerebrospinal fluid (ACSF) containing 4 mM glucose, following 10 min anoxia, the evoked PS did not recover at all after 60 min reoxygenation. When superfusion ACSF contained 10 mM glucose with or without 0.5 mM alpha-cyano-4-hydroxycinnate (4-CIN), after 60 min reoxygenation the evoked PS completely recovered following 10 min anoxia. When superfusion ACSF contained 20 mM glucose with or without 1 mM sodium cyanide (NaCN), after 60 min reoxygenation the evoked PS completely recovered even following 120 min anoxia. In contrast, when superfusion ACSF contained 4 mM glucose, following 10 min 1 mM NaCN chemical anoxia alone, without anoxic anoxia, the evoked PS displayed no recovery after 60 min reoxygenation. Moreover, when 16 mM mannitol and 16 sodium L-lactate were added into 4 mM glucose ACSF, following 10 min anoxia the evoked PS failed to recover at all after 60 min reoxygenation. The results indicate that elevated glucose concentration powerfully protected the synaptic transmission against anoxic damage, and the powerful protection is due to anaerobic metabolism of glucose and not a result of the higher osmolality in higher glucose ACSF. We conclude that lack of glucose is the major cause of anoxia-induced synaptic transmission damage, and that if sufficient glucose is supplied, glycolysis could prevent this damage in vitro.     

Changes in microtubule turnover accompany synaptic plasticity and memory formation in response to contextual fear conditioning in mice

Synaptic plasticity plays a crucial role in learning, memory, and cognitive disorders. Cytoskeletal reorganization underlies neuronal synaptic plasticity, but little is known about the regulation of cytoskeletal dynamics in living animals. We used stable isotope labeling to measure the turnover of tubulin in defined microtubule (MT) populations in murine brain. Neuronal MTs generally exhibited low turnover rates in vivo. Basal turnover was highest in tau-associated MTs, intermediate in microtubule-associated protein 2 (MAP2)–associated MTs, and lowest in cold-stable MTs. Labeling of MTs in mature neurons in cell culture yielded similar turnover results. Intracerebroventricular glutamate injection stimulated, via N-methyl-d-aspartic acid receptors, label incorporation (turnover) in cold-stable, tau-associated, and MAP2-associated MTs, the last of which was shown to be dependent on cyclic adenosine-3′, 5′-monophosphorothioate–protein kinase A. Contextual fear conditioning, a hippocampus-mediated form of memory formation, was accompanied by increased turnover of hippocampal MAP2-associated and cold-stable MTs. Treatment with the MT-depolymerizing drug nocodazole reversed the conditioning-induced increase in label incorporation in MAP2-associated MTs, reduced dendritic spine density, and impaired memory formation. The effects of nocodazole on MT turnover were prevented by the MT-stabilizing agent Taxol (Sigma-Aldrich, St. Louis, MO, USA) and by brain-derived nerve growth factor, both of which also restored dendritic spine density and memory formation in this model. In conclusion, these results suggest that changes in hippocampal MT turnover are required for, and are a biomarker of, the synaptic plasticity that is involved in memory formation.     

Trace fear conditioning enhances synaptic and intrinsic plasticity in rat hippocampus

Experience-dependent synaptic and intrinsic plasticity are thought to be important substrates for learning-related changes in behavior. The present study combined trace fear conditioning with both extracellular and intracellular hippocampal recordings to study learning-related synaptic and intrinsic plasticity. Rats received one session of trace fear conditioning, followed by a brief conditioned stimulus (CS) test the next day. To relate behavioral performance with measures of hippocampal CA1 physiology, brain slices were prepared within 1 h of the CS test. In trace-conditioned rats, both synaptic plasticity and intrinsic excitability were significantly correlated with behavior such that better learning corresponded with enhanced long-term potentiation (LTP; r = 0.64, P < 0.05) and a smaller postburst afterhyperpolarization (AHP; r = -0.62, P < 0.05). Such correlations were not observed in pseudoconditioned rats, whose physiological data were comparable to those of poor learners and naive and chamber-exposed control rats. In addition, acquisition of trace fear conditioning did not enhance basal synaptic responses. Thus these data suggest that within the hippocampus both synaptic and intrinsic mechanisms are involved in the acquisition of trace fear conditioning.     

Tumor necrosis factor alters synaptic transmission in rat hippocampal slices.

The effects of human recombinant tumor necrosis factor (TNF-alpha) on the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. Population spikes and/or excitatory postsynaptic potentials were extracellularly recorded in hippocampus CA1 region from stratum pyramidale and stratum radiatum, respectively, and synaptic transmission was examined in the Schaffer collateral/commissural-CA1 pathway. Basal neurotransmission slightly and promptly increased in slices acutely exposed to TNF-alpha (1-100 nM). Examination of the long-term potentiation (LTP) revealed that a brief treatment with the cytokine did not influence LTP, while a long-lasting application of TNF-alpha (50 min or more) inhibited LTP in a dose-dependent way in the range of 1-100 nM. A role for TNF-alpha as a peptide of immunological significance belonging to the family of brain neuromodulators is discussed.     

Effects of exercise and environmental complexity on deficits in trace and contextual fear conditioning produced by neonatal alcohol exposure in rats

In rodents, voluntary exercise and environmental complexity increases hippocampal neurogenesis and reverses spatial learning and long‐term potentiation deficits in animals prenatally exposed to alcohol. The present experiment extended these findings to neonatal alcohol exposure and to delay, trace, and contextual fear conditioning. Rats were administered either 5.25 g/kg/day alcohol via gastric intubation or received sham‐intubations (SI) between Postnatal Day (PD) 4 and 9 followed by either free access to a running wheel on PD 30–41 and housing in a complex environment on PD 42–72 (wheel‐running plus environmental complexity; WREC) or conventional social housing (SHSH) from PD 30 to 72. Adult rats (PD 80 ± 5) received 5 trials/day of a 10‐s flashing‐light conditioned stimulus (CS) paired with .8 mA footshock either immediately (delay conditioning) or after a 10‐s trace interval (trace conditioning) for 2 days. Neonatal alcohol exposure impaired context and trace conditioning, but not short‐delay conditioning. The WREC intervention did not reverse these deficits, despite increasing context‐related freezing in ethanol‐exposed and SI animals. © 2012 Wiley Periodicals, Inc. Dev Psychobiol 55: 483–495, 2013     

Sex differences in contextual fear conditioning are associated with differential ventral hippocampal extracellular signal-regulated kinase activation

Although sex differences have been reported in hippocampal-dependent learning and memory, including contextual fear memories, the underlying molecular mechanisms contributing to such differences are not well understood. The present study examined the extent to which sex differences in contextual fear conditioning are related to differential activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), a protein kinase critically involved in memory formation. We first show that male rats exhibit more long-term retention of contextual fear conditioning than female rats. During a tone test, females spent more time freezing than males, although both sexes exhibited robust retention of auditory fear learning. Using Western blot analysis, we then show that phosphorylated ERK levels in ventral, but not dorsal, hippocampus are higher in males than females, relative to same-sex controls, 60 minutes after fear conditioning. Post-conditioning increases in ERK activation were observed in the amygdala in both males and females, suggesting a selective effect of sex on hippocampal ERK activation. Together, these findings suggest that differential activation of the ERK signal transduction pathway in male and female rats, particularly in the ventral hippocampus, is associated with sex differences in contextual fear.     

Effects of psilocybin on hippocampal neurogenesis and extinction of trace fear conditioning

Drugs that modulate serotonin (5-HT) synaptic concentrations impact neurogenesis and hippocampal (HPC)-dependent learning. The primary objective is to determine the extent to which psilocybin (PSOP) modulates neurogenesis and thereby affects acquisition and extinction of HPC-dependent trace fear conditioning. PSOP, the 5-HT_2A agonist 25I-NBMeO and the 5-HT_2A/C antagonist ketanserin were administered via an acute intraperitoneal injection to mice. Trace fear conditioning was measured as the amount of time spent immobile in the presence of the conditioned stimulus (CS, auditory tone), trace (silent interval) and post-trace interval over 10 trials. Extinction was determined by the number of trials required to resume mobility during CS, trace and post-trace when the shock was not delivered. Neurogenesis was determined by unbiased counts of cells in the dentate gyrus of the HPC birth-dated with BrdU co-expressing a neuronal marker. Mice treated with a range of doses of PSOP acquired a robust conditioned fear response. Mice injected with low doses of PSOP extinguished cued fear conditioning significantly more rapidly than high-dose PSOP or saline-treated mice. Injection of PSOP, 25I-NBMeO or ketanserin resulted in significant dose-dependent decreases in number of newborn neurons in hippocampus. At the low doses of PSOP that enhanced extinction, neurogenesis was not decreased, but rather tended toward an increase. Extinction of “fear conditioning” may be mediated by actions of the drugs at sites other than hippocampus such as the amygdala, which is known to mediate the perception of fear. Another caveat is that PSOP is not purely selective for 5-HT_2A receptors. PSOP facilitates extinction of the classically conditioned fear response, and this, and similar agents, should be explored as potential treatments for post-traumatic stress disorder and related conditions.     

Effects of lesions to the hippocampus on contextual fear: evidence for a disruption of freezing and avoidance behavior but not context conditioning.

The effects of ibotenic lesions of the hippocampus on conditioning to contextual cues during classical fear conditioning in rats were evaluated by (a) the amount of freezing elicited by contextual cues and (b) the relative avoidance of a shock compartment. In Experiment 1, lesions to the hippocampus had no effect on contextual freezing and marginally affected avoidance after repeated sessions. Experiment 2 showed that lesions to the hippocampus disrupted avoidance when tested after a single conditioning session, while leaving unaffected the acquisition of contextual freezing. Experiment 3 indicated that these lesions decreased the acquisition of contextual freezing when higher footshock intensity was used but had no effect on avoidance after repeated conditioning sessions. These results show that freezing and avoidance do not quantify context conditioning similarly. They further indicate that lesions to the hippocampus may disrupt the expression of these behaviors used as measures of context conditioning but not the acquisition of context conditioning per se.     

Decremental effects of context exposure following delay eyeblink conditioning in rabbits

The conditioning context arises from the relatively static features of the training environment. In rabbit eyeblink conditioning, procedures that retard acquisition (conditioned stimulus [CS] preexposure, unconditioned stimulus preexposure, blocking manipulations) are attenuated by context changes. In this article the authors investigate the effect of context exposure after initial delay conditioning. After conditioned responses (CRs) were established, one group received 6 sessions of context exposure, whereas control groups either remained in their home cages or received exposure to handling and a novel context. Thereafter, all groups received CS-alone testing. The expression of CRs was substantially reduced following context exposure relative to any retention loss in the home-cage control. Exposure to handling and a novel context facilitated the CRs rather than reducing them. ((c) 2006 APA, all rights reserved).     

Characterization of an N-methyl-D-aspartate receptor component of synaptic transmission in rat hippocampal slices

The involvement of N-methyl-D-aspartate receptors in synaptic transmission from Schaffer collateral-commissural fibres to CA1 neurons has been investigated in rat hippocampal slices. When the perfusion medium was changed from one containing 1 mM Mg2+ to one with no added Mg2+ there was a pronounced increase in the amplitude of the population spike, the appearance of secondary population spikes and in some slices spontaneous epileptiform discharges developed. The secondary and spontaneous population spikes were abolished by the selective N-methyl-D-aspartate antagonist, D-2-amino-5-phosphonovalerate. The effects on the primary population spike depended on the strength of synaptic activation. At low intensities, the N-methyl-D-aspartate antagonist reduced or abolished this response whereas at high intensities the primary population spike was slightly increased in amplitude by this compound. Mg2+ had dose-dependent (20-500 microM) effects on synaptic responses which were identical to those of D-2-amino-5-phosphonovalerate. Increasing the Ca2+ concentration over a range of 1-3 mM also reduced or abolished secondary population spikes and, at low stimulus intensities, the primary population spike. At higher stimulus intensities, however, the primary population spike was insensitive to the Ca2+ concentration over this range. These results demonstrate the major extent to which N-methyl-D-aspartate receptors can contribute to synaptic transmission and epileptiform activity in the CA1 region of the hippocampus. They also show that an important role of Mg2+ in this region is to prevent significant activation of this receptor system during low-frequency synaptic transmission.     

Effects of the anticonvulsant lamotrigine and carbamazepine on synaptic transmission in the CA1 area of rat hippocampal slices

Lamotrigine and carbamazepine (10⁻³ M) almost completely inhibit electrical activity of neurons in hippocampal slices. The effect of these drugs on signal transmission in the system of Schaffer collaterals/commissural fibers—CA1 hippocampal area pyramidal neurons shows that both anticonvulsants inhibit the excitability of the presynaptic axons and transmission efficiency in the glutamatergic synapses without any significant influence on signal transmission from a synapse to the spike generator region in the postsynaptic pyramidal neurons. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 9, pp. 307–310, September, 1998     

Slow synaptic inhibition in relation to frequency habituation in dentate granule cells of rat hippocampal slices

Summary In paired pulse stimulation experiments the mechanism underlying frequency habituation of postsynaptic potentials in dentate granule cells of rat hippocampal slices was studied by measuring extra and intracellular potentials as well as changes in extracellular calcium ([Ca²⁺]₀) and potassium concentrations ([K⁺]₀). Orthodromic stimulation of the perforant path induced in most granule cells a late, slow hyperpolarization (SH), lasting for up to 1.2 s. During the SH the membrane conductance was increased by up to 40%. The reversal potential of the SH was around -90 mV and varied with the [K⁺]₀. Frequency habituation was seen in all cells with the SH, whereas cells which display frequency potentiation had no SH. Lowering of [Ca²⁺]₀ reversed paired pulse induced frequency habituation into frequency potentiation at [Ca²⁺]₀ levels where the SH disappeared. Phaclofen blocked the SH and reversed frequency habituation into frequency potentiation. Elevating [Mg²⁺]₀ also reversed frequency habituation into frequency potentiation and reduced the SH. We conclude that the SH represents a late, slow IPSP which is responsible for frequency habituation in dentate granule cells. We noted that during repetitive stimulation the SH soon started to fade. This effect can in part be attributed to extracellular K⁺-accumulation as suggested by the K⁺-dependence of the slow IPSP and the observations of changes in [K⁺]₀ during repetitive stimulation. This could explain why frequency habituation reverses into frequency potentiation during repetitive stimulation.On leave from the Department of Pharmacology, Uniformed Services, University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA     

Whole-cell patch-clamp recordings of an NMDA receptor-mediated synaptic current in rat hippocampal slices

Whole-cell patch-clamp recordings and pharmacological techniques have been used to obtain low noise recordings of 2 excitatory postsynaptic synaptic currents (termed EPSCA and EPSCB) evoked by stimulation of the Schaffer collateral-commissural pathway in rat hippocampal slices. EPSCA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and EPSCB was blocked by D-2-amino-5-phosphonovalerate (APV), indicating their mediation by non-N-methyl-D-aspartate (non-NMDA) and NMDA receptors, respectively. EPSCB has a slower time-course than EPSCA and its current-voltage relationship was highly non-linear with a region of negative slope conductance from -35 to -100 mV. These properties of EPSCA and EPSCB can explain their differing participation in synaptic transmission in this pathway.