Induction of mossy fiber --> Ca3 long-term potentiation requires translocation of synaptically released Zn2+.

The mammalian CNS contains an abundance of chelatable Zn(2+) sequestered in the vesicles of glutamatergic terminals. These vesicles are particularly numerous in hippocampal mossy fiber synapses of the hilar and CA3 regions. Our recent observation of frequency-dependent Zn(2+) release from mossy fiber synaptic terminals and subsequent entry into postsynaptic neurons has prompted us to investigate the role of synaptically released Zn(2+) in the induction of long-term potentiation (LTP) in field CA3 of the hippocampus. The rapid removal of synaptically released Zn(2+) with the membrane-impermeable Zn(2+) chelator CaEDTA (10 mm) blocked induction of NMDA receptor-independent mossy fiber LTP by high-frequency electrical stimulation (HFS) in rat hippocampal slices. Mimicking Zn(2+) release by bath application of Zn(2+) (50-100 microm) without HFS induced a long-lasting potentiation of synaptic transmission that lasted more than 3 hr. Moreover, our experiments indicate the effects of Zn(2+) were not attributable to its interaction with extracellular membrane proteins but required its entry into presynaptic or postsynaptic neurons. Co-released glutamate is also essential for induction of LTP under physiological conditions, in part because it allows Zn(2+) entry into postsynaptic neurons. These results indicate that synaptically released Zn(2+), acting as a second messenger, is necessary for the induction of LTP at mossy fiber-->CA3 synapses of hippocampus.

Unilateral LTP triggers bilateral increases in hippocampal neurotrophin and trk receptor mRNA expression in behaving rats: evidence for interhemispheric communication.

Induction of long-term potentiation (LTP) in the dentate gyrus of awake rats triggered a rapid (2 hour) elevation in tyrosine kinase receptor (trkB and trkC) gene expression and a delayed (6-24 hour) increase in brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) gene expression. Depending on the mRNA species, LTP induction led to highly selective unilateral or bilateral increases in gene expression. Specifically, trkB and NT-3 mRNA elevations were restricted to granule cells in the ipsilateral dentate gyrus, whereas bilateral increases in trkC, BDNF, and nerve growth factor (NGF) mRNA levels occurred in granule cells and hippocampal pyramidal cells. Both unilateral and bilateral changes in gene expression were N-methyl-D-aspartate (NMDA) receptor-dependent and LTP-specific. Bilateral electrophysiological recordings demonstrated that LTP was unilaterally induced; this was corroborated by a dramatic unilateral increase in the expression of the immediate early gene zif/268, a marker for LTP, restricted to the ipsilateral granule cells. The results indicate that LTP triggers an interhemispheric communication manifested as selective, bilateral increases in gene expression at multiple sites in the hippocampal network. Furthermore, our findings suggest that physiological plastic changes in the adult brain may involve coordinated, time-dependent regulation of multiple neurotrophin and trk receptor genes.

Responses to GABA recorded from identified rat visual cortical neurons.

Responses to GABA were recorded from 87 neurons of rat visual cortical slices. Pyramidal and nonpyramidal cells were identified by intracellular dye injection, and their responses were compared. All identified pyramidal and nonpyramidal cells, as well as unstained cells, responded to GABA ejected from a pipette that was positioned within 300 micron of the soma. Their responses were similar, regardless of their morphology. In addition, GABA responses of visual cortical neurons could not be distinguished from those of other areas of the neocortex, or pyramidal cells in area CA1 of hippocampus. Depending on the site of application, there appeared to be two types of GABA responses that were present in all cells. The first was generated by application of GABA to the soma (GABAs response; mean reversal potential = -71.7 mV). The second occurred when GABA was applied on dendrites (GABAD response; mean reversal potential = -49.3 mV). When GABA was ejected on proximal dendritic regions, both responses could be observed in combination. Both GABAs and GABAD responses were accompanied by extremely large increases in conductance. In some cells, a third type of GABA response was elicited following somatic or dendritic GABA application. This response was a relatively small-amplitude, long-lasting hyperpolarization which followed a GABAS and GABAD response (late hyperpolarization, mean reversal potential = -79.8 mV).

gamma-Aminobutyrate sensitivity does not change during long-term potentiation in rat hippocampal slices.

Long-term potentiation is a long-lasting enhancement of synaptic efficacy following brief, high-frequency, repetitive stimulation of a monosynaptic input. Intracellular recordings have shown that the inhibitory postsynaptic potential changes in amplitude during long-term potentiation. Yet how this may occur is unclear. To test for a possible alteration in postsynaptic sensitivity to the recurrent inhibitory transmitter gamma-aminobutyrate, we have examined the effect of gamma-aminobutyrate, focally applied to the hippocampal CA1 cell-body layer, on the extracellular recorded action potential (population spike). We found that the degree, duration, dose-dependence and time-course of inhibition produced by gamma-aminobutyrate are unchanged during long-term potentiation. This suggests that a change in sensitivity of CA1 pyramidal cells to the transmitter gamma-aminobutyrate is not the reason for the alteration in the inhibitory postsynaptic potential during long-term potentiation.

Characterization of extracellular accumulation of Zn2+ during ischemia and reperfusion of hippocampus slices in rat.

The mammalian CNS contains an abundance of chelatable zinc that is sequestered in the vesicles of glutamatergic presynaptic terminals and co-released with glutamate. Considerable Zn(2+) is also released during cerebral ischemia and reperfusion (I/R) although the mechanism of this release has not been elucidated. We report here the real time observation of increase of the concentration of extracellular Zn(2+) ([Zn(2+)](o)), accompanied by a rapid increase of intracellular free Zn(2+)concentration, in the areas of dentate gyrus (DG), CA1 and CA3 in acute rat hippocampus slices during ischemia simulated by deprivation of oxygen and glucose (OGD) followed by reperfusion with normal artificial cerebrospinal fluid. A brief period of OGD caused a sustained increase of [Zn(2+)](o). Subsequent reperfusion with oxygenated medium containing glucose resulted in a further increase of [Zn(2+)](o). Longer periods of OGD caused greater increases of [Zn(2+)](o,) and subsequent reperfusion caused still further increases of [Zn(2+)](o,) regardless of OGD duration. The Zn(2+) chelator CaEDTA (10 mM) significantly reduced the increase of [Zn(2+)] induced by OGD and reperfusion. Significant regional differences of [Zn(2+)](o) over the areas of the DG, CA1 and CA3 were not observed during I/R. Neither sodium channel blockade by tetrodotoxin (2 microM), perfusion with nominally calcium-free medium nor anatomical disassociation of the DG, CA1 and CA3 regions from one another by lesioning affected the increase of [Zn(2+)](o). The non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine methyl ester (1 mM), however, blocked the increase of [Zn(2+)](o) during ischemia and reperfusion. The data indicate the important role of NO in causing the release of Zn(2+) during I/R and suggest that NOS inhibitors may be used to reduce Zn(2+)-induced neuronal injury.

Muscarinic depression of synaptic transmission and blockade of norepinephrine-induced long-lasting potentiation in the dentate gyrus.

Bath application of the muscarinic receptor agonist, muscarine, produced a concentration-dependent depression of synaptic activity in the dentate gyrus of hippocampal slices. A concentration of 10 microM muscarine produced a reversible depression that could be competitively antagonized by the muscarinic receptor antagonist pirenzepine. However, other muscarinic receptor subtype (M1-M3) antagonists could also block the effects of muscarine. The rank order of antagonist potency was: 4-diphenylacetoxy-N-methyl-piperidine methiodide (M3/M1 antagonist) > pirenzepine (M1) > AFDX-116 (M2). The depression produced by 10 microM muscarine was not affected by in vivo pretreatment with pertussis toxin, and therefore was not mediated by a pertussis toxin-sensitive G-protein. In addition, high concentrations of muscarine did not affect either basal or isoproterenol-stimulated accumulation of cyclic AMP from slices of dentate gyrus. Muscarine also produced a concentration-dependent blockade of the induction of norepinephrine-induced long-lasting potentiation in the dentate gyrus. Norepinephrine-induced long-lasting potentiation is a form of long-lasting plasticity induced in medial perforant path synapses by beta-adrenergic agonists such as isoproterenol. The muscarinic blockade of norepinephrine-induced long-lasting potentiation was also prevented by pretreatment with pirenzepine. Based on these pharmacological data, we conclude that muscarinic depression of evoked responses, as well as blockade of norepinephrine-induced long-lasting potentiation, involves activation of either M3 or M1, but not M2, muscarinic receptors. These data also demonstrate that in addition to modulating normal synaptic transmission, muscarinic receptors may also play an important role in modulating synaptic plasticity.

Free radicals accelerate the decay of long-term potentiation in field CA1 of guinea-pig hippocampus.

Free radicals have been implicated in a number of pathological conditions. To evaluate the neurophysiological consequences of free radical exposure, slices of hippocampus isolated from guinea-pigs were exposed to hydrogen peroxide which reacts with tissue iron to generate hydroxyl free radicals. Long-term potentiation, a sustained increase in synaptic responses, was elicited in field CA1 by high frequency stimulation of an afferent pathway. We found that 0.002% peroxide did not directly affect the responses evoked by stimulation of the afferent pathway but did prevent maintenance of long-term potentiation. Short-term potentiation and paired-pulse facilitation were not affected by peroxide treatment. Peroxide was less effective if removed following high frequency stimulation and was ineffective if applied only after high frequency stimulation. Input/output analysis showed that the increase in synaptic efficacy was reduced with peroxide treatment. Changes in the enhanced ability of the synaptic potential to generate a spike were less apparent. These data show that the interference of free radicals with long-term potentiation may contribute to pathological deficits. It is possible that intracellular calcium regulation is disrupted by peroxide treatment. A number of second messenger systems involved with long-term potentiation are potential targets for free radical attack.

LTP in the lateral perforant path is beta-adrenergic receptor-dependent.

Norepinephrine induces an activity-independent long-lasting depression of synaptic transmission in the lateral perforant path input to dentate granule cells, whereas high frequency stimulation induces activity-dependent long-term potentiation (LTP). We investigated the role of endogenous activation of beta-adrenergic receptors in LTP of the lateral and medial perforant paths under conditions affording selective stimulation of these pathways in the rat hippo-campal slice. Propranolol (1 microM), a beta-receptor antagonist, blocked LTP induction of both lateral and medial perforant path-evoked field excitatory postsynaptic potentials. The results indicate a broad requirement for norepinephrine in different types of synaptic plasticity, including activity-independent depression and activity-dependent LTP in the lateral perforant path.

Long-term potentiation: studies in the hippocampal slice.

Long-term potentiation (LTP) is an example of activity-dependent plasticity that was discovered in the hippocampal formation. There is growing evidence that LTP not only is a useful model for mnemonic processes, but also may represent the cellular substrate for at least some kinds of learning and memory. The hippocampal slice preparation has proven exceptionally useful in pharmacological studies of possible mechanisms of LTP. A slice remains viable and stable for several hours, and known concentrations of drugs in the bathing medium can be added and then washed out. Drugs can also be applied under visual guidance from micropipettes to discrete neuronal regions, an accomplishment that is aided by the lamellar organization of the hippocampus. Electrical stimulation of the perforant path (PP) in the molecular layer of the dentate gyrus produces a monosynaptic excitatory postsynaptic potential (EPSP) and action potential, which can be recorded extracellularly as a population EPSP and population spike, respectively. Presentation of a high-frequency train (HFT; 100 Hz X 1 s) to the PP results in a long-lasting (greater than 30 min) potentiation of the maximal EPSP slope and of the population spike amplitude. Similarly, exposure of the slice to norepinephrine (e.g. 20 microM for 30 min) results in a long-lasting potentiation (LLP) of both EPSP and population spike (Stanton and Sarvey (1987) Brain Res. Bull., 18: 115). No such LLP was seen in field CA1 following NE application (Stanton and Sarvey (1985) Brain Res., 361: 276). beta-Adrenergic antagonists, such as propranolol, inhibit both LTP and NE-induced LLP in dentate (Stanton and Sarvey, J. Neurosci., 5: 2169 (1985); Stanton and Sarvey (1985) Brain Res., 361: 276). Cyclic AMP levels are increased by either an HFT or NE (Stanton and Sarvey (1985) Brain Res., 358: 343). Thus, NE, acting through a beta-receptor, appears to be both necessary and sufficient to produce long-lasting enhancement of synaptic responses. Finally, inhibitors of protein synthesis, such as emetine, also block both LTP and NE-induced LLP (Stanton and Sarvey, J. Neurosci., (1984) 4: 3080; Stanton and Sarvey (1985) Brain Res., 361: 276). The N-methyl-D-aspartate (NMDA) excitatory amino acid receptor subtype appears to play a role in a number of forms of neuronal plasticity. Bath-application of a 1 microM concentration of the NMDA antagonists D-2-amino-5-phosphonavaleric acid (AVP) or 3-((+/-)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) blocked both LTP and NE-induced LLP in the dentate gyrus.(ABSTRACT TRUNCATED AT 400 WORDS)

Postsynaptic firing during repetitive stimulation is required for long-term potentiation in hippocampus.

Long-term potentiation (LTP) in the hippocampus is a long lasting enhancement of the postsynaptic evoked response following high frequency, repetitive stimulation of afferents. The extracellularly recorded action potential (population spike) can be reversibly blocked, without affecting the extracellularly recorded excitatory postsynaptic potential, by focal application of gamma-aminobutyric acid, tetrodotoxin, or pentobarbital, to the CA1 pyramidal cells of the hippocampal slice. When the population spike is blocked during repetitive stimulation, LTP does not occur. It appears that postsynaptic firing of action potentials during repetitive stimulation is necessary to produce LTP.

Beta-adrenergic agonist-induced long-lasting synaptic modifications in hippocampal dentate gyrus require activation of NMDA receptors, but not electrical activation of afferents.

Isoproterenol induced long-lasting potentiation (LLP) of the medial perforant path-evoked excitatory post-synaptic potential (EPSP) and long-lasting depression (LLD) of the lateral perforant path-evoked EPSP in the absence of perforant path activation. The NMDA receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid [D(-)APV] blocked the induction of LLP and LLD. After wash, a subsequent exposure to isoproterenol induced only LLP of medial perforant path EPSPs; LLD of lateral perforant path-evoked EPSPs did not occur. Our results are consistent with the hypothesis that beta-adrenergic agonist-induced synaptic modifications in the dentate gyrus arise from pre- and postsynaptic events.

The effect of high-frequency electrical stimulation and norepinephrine on cyclic AMP levels in normal versus norepinephrine-depleted rat hippocampal slices.

Cyclic 3',5'-adenosine monophosphate (cAMP) generation by neuronal activity and norepinephrine (NE) was studied in rat hippocampal slices. High-frequency perforant path stimulation increased cAMP levels 2.5-fold in the dentate gyrus 1 min, but not 30 min, post-stimulation. This increase was abolished by depletion of NE with 6-hydroxydopamine. NE (50 microM) also caused a 3-fold rise in cAMP in whole slices and this stimulation was not altered by NE depletion. These results are consistent with our previous data suggesting that cAMP production is involved in the expression of long-term potentiation and NE-induced long-lasting potentiation in the dentate gyrus.

Blockade of norepinephrine-induced long-lasting potentiation in the hippocampal dentate gyrus by an inhibitor of protein synthesis.

The mechanism of action of norepinephrine (NE)-induced potentiation of the population spike in the dentate gyrus of hippocampal slices was examined and compared with NE effects in field CA1. NE-induced potentiation was confined to the dentate gyrus, where slices perfused for 30 min with concentrations of NE as low as 5 microM exhibited potentiation of the perforant path evoked population spike. Potentiation began within 15 min, and lasted many hours after NE was washed out. Experiments where slices were pre-incubated with the protein synthesis inhibitor emetine indicated that there are two distinct phases to NE-induced potentiation. The initial short-term NE-induced potentiation (NEP) seen during NE application was not affected by a 30 min pre-incubation with emetine, whereas the long-lasting potentiation (NELLP) which persists after NE washout was completely blocked by emetine at a concentration which we have previously shown to be effective in blocking hippocampal long-term potentiation (LTP). Additional experiments indicated that both phases of NE-induced potentiation were completely blocked by the beta-antagonist propranolol and the beta 1-antagonist metoprolol. Furthermore, pre-incubation of slices with the direct-acting adenylate cyclase stimulant forskolin shifted the dose-response curves for both phases of NE-induced potentiation to the left. These results suggest that NE-induced potentiation is probably mediated by beta 1-receptor stimulation of adenylate cyclase. We have previously shown an importance for beta 1-receptor stimulation of adenylate cyclase in the production of LTP in the dentate. Thus, these results demonstrate a number of similarities between hippocampal LTP and NELLP in the dentate gyrus.

Responses to gamma-aminobutyric acid applied to cell bodies and dendrites of rat visual cortical neurons.

The effects of pressure-applied gamma-aminobutyric acid (GABA) on the soma and dendrites of pyramidal and non-pyramidal neurons of rat visual cortical slices were recorded intracellularly. When applied close to the soma, GABA produced hyperpolarizations and depolarizations, but when GABA was applied more than 250 microns from the soma only depolarizations were recorded. The results suggest that most visual cortical cells respond to GABA and that the responses of pyramidal and non-pyramidal cells to GABA are similar.

NMDA receptor antagonists block norepinephrine-induced long-lasting potentiation and long-term potentiation in rat dentate gyrus.

In the in vitro rat dentate gyrus, norepinephrine-induced long-lasting potentiation (NELLP) and long-term potentiation (LTP) of responses to perforant path stimulation were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D(-)APV) and 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP). CPP and D(-)APV, but not L(+)APV, also depressed the orthodromic population spike but not the antidromic spike, which suggests that these receptors may function in low-frequency evoked activity of granule cells. We conclude that NELLP, like LTP in the dentate gyrus, requires NMDA receptor activation.

Acute cold stress leading to elevated corticosterone neither enhances synaptic efficacy nor impairs LTP in the dentate gyrus of freely moving rats.

Exposure to stress has previously been found to impair long-term potentiation (LTP) in the hippocampus. Exposure to stress has also been proposed to induce an LTP-like effect. We examined the effect of acute cold stress on synaptic transmission, neuronal excitability, and LTP induction in the medial perforant path-granule cell synapse of freely moving rats. After obtaining baseline recordings of evoked field potentials at room temperature (23 degrees C), rats were transferred to an environmental cage maintained at 4 degrees C (cold group) or 23 degrees C (control group) and, 90 min later, high-frequency stimulation (HFS) was applied to the medial perforant path. Serum corticosterone measured in trunk blood from rats without implanted electrodes was significantly elevated in cold exposed (28. 7 microg/dl) rats relative to control (6.6 microg/dl). Despite increased corticosterone levels indicative of stress activation, cold exposed rats exhibited LTP of the fEPSP slope and population spike of similar magnitude and time course as controls. In addition, there was no stress-specific effect on the fEPSP slope or population spike and no effect on paired-pulse plasticity. Surprisingly, despite extensive cage acclimation, transferring rats to the environmental cage was associated with a reduction in population spike amplitude and an enhancement in paired-pulse facilitation. The results show that acute cold stress leading to elevated serum corticosterone levels neither induces LTP-like increases in synaptic efficacy nor impairs tetanus-evoked LTP in the dentate gyrus of freely moving rats. Thus, impaired working memory during cold stress is not due to an inability of perforant path synapses to express LTP.

Muscarinic receptor activation facilitates the induction of long-term potentiation (LTP) in the rat dentate gyrus.

Bath application of two different concentrations of muscarine produced two different effects on evoked responses in the dentate gyrus of rat hippocampal slices. A concentration of 1 microM muscarine did not affect the evoked population spike or excitatory postsynaptic potential (EPSP), but facilitated the induction of LTP. In contrast, a concentration of 10 microM muscarine depressed both the population spike and EPSP, but had no effect on LTP induction. The M1 muscarinic receptor antagonist pirenzepine (1 microM) blocked the muscarine-induced facilitation of LTP, but had no effect on the depression of evoked responses. These data suggest that activation of M1 receptors can facilitate the induction of LTP.

Lowering extracellular calcium reverses paired pulse habituation into facilitation in dentate granule cells and removes a late IPSP.

Simultaneous measurements of cellular responses and extracellular field potentials as well as extracellular Ca2+ concentration [( Ca2+]0) were performed in the dentate gyrus granule cell layer during paired pulse stimulation of the lateral perforant path at resting [Ca2+]o and during washout of calcium. At resting [Ca2+]o the second response to a paired stimulus was smaller than the first response. This frequency habituation reversed into frequency potentiation (second response larger than the first one) during lowering of [Ca2+]o at about the same time when a late presumed inhibitory postsynaptic potential (IPSP) was abolished. This suggests that a slow inhibition can account for part of frequency habituation in the dentate gyrus.

Norepinephrine regulates long-term potentiation of both the population spike and dendritic EPSP in hippocampal dentate gyrus.

Hippocampal slices from norepinephrine (NE)-depleted rats exhibited marked reductions in long-term potentiation (LTP) of both the population spike and dendritic EPSP in the dentate gyrus. In contrast, depletion of serotonin (5-hydroxytryptamine, 5-HT) had no effect on either population spike or EPSP-LTP. In addition, superfusion of slices with NE produced potentiation of both the granule cell population spike and dendritic EPSP which persisted long after NE washout. These data support a role for NE in regulating long-term plasticity of both granule cell action potential firing and dendritic EPSPs.

Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion.

"Free Zn2+" (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or "tonic" level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5-25 nM). This concentration is 15,000-fold lower than the "300 microM" concentration which is often used as the "physiological" concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.