Marcello Brunelli

Long-lasting hippocampal potentiation and contextual memory consolidation

In order to ascertain whether there are hippocampal electrophysiological modifications specifically related to memory, exploratory activity and emotional stress, extracellular electrical activity was recorded in hippocampal slices prepared from the brains of male adult rats. Several groups of animals were employed: (i) rats which had freely explored the experimental apparatus (8 min exposure); (ii) rats which had been subjected, in the same apparatus, to a fear conditioning paradigm training entailing the administration of aversive electrical footshocks (8 min exposure); (iii) rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make difficult the association between painful stimuli and the apparatus (30 s exposure); (iv) naïve rats never placed in the apparatus. Half of the rats from each treatment group were used for retrieval testing and the other half for hippocampal excitability testing. The conditioned freezing response was exhibited for no less than 4 weeks. Hippocampal excitability was measured by means of input–output curves (IOC) and paired‐pulse facilitation curves (PPF). Retrieval testing or brain slices preparation were performed at increasing delays after the training sessions: immediately afterwards or after 1, 7 or 28 days. Only the rats subjected to the fear conditioning training exhibited freezing when placed again in the apparatus (retrieval testing). It was found that IOCs, with respect to naïve rats, increased in the conditioned animals up to the 7‐day delay. In free exploration animals the IOCs increased only immediately after the training session. In all other rats no modification of the curves was observed. IOC increases do not appear to imply presynaptic transmitter release modifications, because they were not accompanied by PPF modifications. In conclusion, a clear‐cut correlation was found between the increase in excitability of the Schaffer collateral–CA1 dendrite synapses and freezing response consolidation.

Time‐dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat

The effects of contextual fear‐learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one‐trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear‐learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short‐term (STP) and long‐term potentiation (LTP) when compared to control ‘naïve’ subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long‐term but not short‐term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from ‘shock‐only’ rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings – no conditioned freezing during retention testing). In ‘exploration’ preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired‐pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animals prior experience.

Long‐term Potentiation of NMDA Receptor‐mediated EPSP in Guinea‐pig Hippocampal Slices

Hippocampal slices from guinea‐pigs were used to examine the long‐term potentiation (LTP) of the N‐methyl‐D‐aspartate (NMDA)‐mediated excitatory postsynaptic potential (EPSP). lntracellular recordings were performed from CA1 pyramidal neurons in the presence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5–10 μM) and picrotoxin (50 μM). In these experimental conditions test stimuli applied at low frequency (0.1 Hz) to the Schaffer collateral ‐ commissural pathway evoked a prolonged EPSP (150–200 ms). To obtain this CNQX‐resistant EPSP, stimulus intensities had to be raised above the level required to evoke an EPSP of comparable amplitude in physiological solution. Tetanic stimulation (two trains of 100 Hz, 1 s every 20 s) led to a potentiation of the CNQX‐resistant EPSP, and this potentiated response was abolished with d‐(‐)‐2‐amino‐5‐phosphonovaleric acid (50μM). The potentiation of the NMDA receptor‐mediated EPSP was more pronounced for strong than for weak test stimuli, and was suppressed when test EPSPs were evoked during membrane hyperpolarization. These results suggest that NMDA receptor‐mediated responses can undergo LTP, and hence can contribute to the maintenance of LTP.

Role of serotonin and cyclic AMP on facilitation of the fast conducting system activity in the leechHirudo Medicinalis

In the nervous system of the leech Hirudo medicinalis it has been possible to study short-term plastic changes. Depression and facilitation have been demonstrated in the fast conducting system (FCS) activity; this pathway consists of a chain of electrically linked neurons present in each ganglion. In semi-intact animals or in preparation of nerve cord and segments of body wall, both electrical stimulation of peripheral roots and tactile stimulation of the skin induced, after repetitive stimulation (0.1/s) a prolonged decrement of FCS response. Strong nociceptive stimulation applied onto the head or the body wall produced a sustained facilitation of the waned response. The same potentiation has been observed by perfusing the isolated ganglion with serotonin (5 x 10(-5) M). Such a potentiation is abolished by preincubation with methysergide, an antagonist of serotonin, and with imidazole, a cAMP-phosphodiesterase activator. Such an effect is mimicked by an analog of cAMP, db-cAMP. Simultaneous recordings of both T neurons (intracellularly) and FCS firing discharge showed that, during FCS response decrement, the T cell activity remained unchanged and no modification of conductance occurred, excluding therefore a detectable involvement of sensory neurons in the depression. These results suggest that short-term plastic changes of the FCS of the leech are due to a prolonged potentiation of synaptic transmission as a result of serotonin-mediated increase in cAMP.

The transient depression of hippocampal CA1 LTP induced by chronic intermittent ethanol exposure is associated with an inhibition of the MAP kinase pathway

Using electrophysiological and biochemical approaches, we investigated the effects of chronic, intermittent ethanol (CIE) treatment on activation of the mitogen activated protein kinase (MAPK), also known as extracellular signal regulated protein kinase 1 and 2. In hippocampal slices taken from control rats, brief high‐frequency stimulation to Schaffer collateral fibers induced a large post‐tetanic potentiation (PTP) in the CA1 region that decayed to stable long‐term potentiation (LTP) of field extracellular postsynaptic potentials. Western blot analyses showed that phosphorylation of MAPK was increased during PTP and returned to baseline levels during LTP. In slices from the rats removed immediately from CIE treatment, PTP and MAPK activation during the PTP was significantly less than that observed in control slices and LTP was absent. In slices from rats subjected to 1 day withdrawal from CIE treatment, both the reduction in MAPK phosphorylation during PTP and the impairment of PTP and LTP were still evident. Recovery of PTP and partial recovery of LTP was observed in slices obtained from 5‐day withdrawn rats. However, MAPK activation during PTP was still attenuated significantly. Interestingly, MAPK activation was enhanced significantly during LTP in 5‐day withdrawn rats as well as the sensitivity to MAPK inhibitor PD 098059. In addition to these changes in HFS‐induced MAPK activation, we also observed a significant reduction in the basal phosphorylation of MAPK in slices removed from rats immediately after CIE treatment. These results implicate the MAPK signal transduction pathway as a potential cellular target of ethanol. Alterations in MAPKs could play an important role in the alcohol‐induced changes in synaptic plasticity associated with the effects of alcohol abuse on learning and memory processes.

Effects of dopamine, D-1 and D-2 dopaminergic agonists on the excitability of hippocampal CA1 pyramidal cells in guinea pig

SummaryIn hippocampal pyramidal cells (HPCs), Dopamine (DA) application (1 μM) produced, in 50% of recorded cells, an hyperpolarization of the resting membrane potential (r.m.p.) and an increase of the afterhyperpolarization (AHP) amplitude and duration in 79% of recorded cells. DA-induced effects on both the r.m.p. and AHP were mimicked by bath application of a D-l selective agonist, SKF 38393 (20 μM). In addition, we have observed that a D-l selective antagonist such as SCH 23390 (1 μ,M) abolished the action of both DA and SKF 38393. In contrast, the activation of D-2 receptors through LY 171555 (10 μm) produced, in 50% of cells, a depolarization of the r.m.p. and a depression of the AHP in 67% of recorded cells. These results suggest that the effects observed in hippocampal pyramidal neurons after DA application of micromolar concentration are mediated by D-1 subtype of receptors.

Inhibition of Na+/K+ ATPase potentiates synaptic transmission in tactile sensory neurons of the leech

Increasing evidence indicates that modulation of Na+/K+ ATPase activity is involved in forms of neuronal and synaptic plasticity. In tactile (T) neurons of the leech Hirudo medicinalis, Na+/K+ ATPase is the main determinant of the afterhyperpolarization (AHP), which characterizes the firing of these mechanosensory neurons. Previously, it has been reported that cAMP (3′,5′‐cyclic adenosine monophosphate), which mediates the effects of serotonin (5HT) in some forms of learning in the leech, negatively modulates Na+/K+ ATPase activity, thereby reducing the AHP amplitude in T neurons. Here, we show that a transient inhibition of Na+/K+ ATPase can affect the synaptic connection between two ipsilateral T neurons. Bath application of 10 nm dihydroouabain (DHO), an ouabain analogue, causes an increase in the amplitude of the synaptic potential (SP) recorded in the postsynaptic element when a test stimulus is applied in the presynaptic neuron. Iontophoretic injection of cAMP into the presynaptic T neuron also produces an increase of SP. Simulations carried out by using a computational model of the T neuron suggest that a reduction of the pump rate and a consequent depression of the AHP might facilitate the conduction of action potentials to the synaptic terminals. Moreover, nearly intact leeches injected with 10 nm DHO respond with a swimming episode more quickly to an electrical stimulation, which selectively activates T neurons exhibiting sensitization of swimming induction. Collectively, our results show that inhibition of Na+/K+ ATPase is critical for short‐term plasticity.

Caulerpenyne, a toxin from the seaweed Caulerpa taxifolia, depresses afterhyperpolarization in invertebrate neurons

The massive invasion of the Mediterranean Sea by the tropical seaweed Caulerpa taxifolia (Vahl) C. Agardh has stimulated several investigations in order to test the environmental risk from an ecotoxicological point of view. The studies carried out on various experimental models have shown that caulerpenyne, the major metabolite synthesized by the seaweed, affects several cellular and molecular targets. In addition, neurological disorders have been reported in patients who accidentally ate C. taxifolia, but no evidence about the potential effects of the seaweed and of its metabolites on nerve cells were up to now available. Herein we describe that caulerpenyne modifies the electrical properties of touch mechanosensory cells of the leech Hirudo medicinalis. The physiological firing of these cells causes an afterhyperpolarization that is mainly due to the activity of the Na+/K+-ATPase and to a lesser extent to a calcium-dependent potassium current. Caulerpenyne depressed this afterhyperpolarization; the effect was dose-dependent and partially reversible. Experiments have been carried out in order to understand the mechanism through which caulerpenyne reduced the afterhyperpolarization. The action of the biotoxin has been tested in the presence of pharmacological blockers of calcium-dependent potassium channels such as cadmium and apamin. In these experimental conditions, caulerpenyne still reduced the residual afterhyperpolarization, suggesting a direct effect of the toxin on the Na+/K+-ATPase. In order to test this hypothesis, we have performed experiments where the Na+/K+-ATPase was activated by the intracellular injection of sodium and where also its basal activity was modified as well. From the data collected we suggest that caulerpenyne inhibits both the basal and the sodium-induced activity of the Na+/K+-ATPase in leech touch neurons.

Increased synthesis of cyclic AMP and short-term plastic changes in the segmental ganglia of the LeechHirudo medicinalis

Summary1.The effects of different neurotransmitters on cAMP levels in segmental ganglia ofHirudo were studied. Serotonin and dopamine caused a significant increase in cAMP levels. Norepinephrine, octopamine, glutamate, and carbachol were ineffective.2.The stimulation of cAMP levels by dopamine was blocked by increasing the concentration of Mg2+ ions in the incubation bath. Serotonin was still effective in stimulating cAMP synthesis in the presence of high Mg2+ concentrations. Imidazole, a cAMP-phosphodiesterase activator, markedly inhibited the increase in cAMP induced by serotonin. Serotonin, but not dopamine, significantly stimulated adenylate cyclase activity in homogenates of segmental ganglia. The serotonin effect was blocked by RMI 12330A.3.Electrical stimulation of peripheral roots induced excitation of the fast conducting system. Repetitive stimulation (0.1/sec) resulted in a waning of firing discharge (habituation). A strong noxious stimulus applied to another pathway gave rise to a potentiation of the depressed response (dishabituation). The dishabituation was abolished by methysergide.4.These results indicate that serotonin may mediate short-term plastic changes such as dishabituation through a mechanism involving cAMP in the leechHirudo.

Serotonin and Retzius cell depress the hyperpolarization following impulses of leech touch cell

Intracellular recordings from T mechanosensory cells of Hirudo medicinalis showed, as previously demonstrated, that repetitive firing is followed by a long-lasting hyperpolarization. Serotonin application at two concentrations (1 microM and 50 microM) depressed this hyperpolarization by up to 2/3; the effect was dose-dependent, long-lasting and reversible. Intracellular stimulation of giant serotonergic neurons (Retzius cells, Rz) mimicked serotonin perfusion: the effect was proportional to the number of spikes fired by Retzius cells. The combined use of intracellular iontophoretic injection of horseradish peroxidase and lucifer yellow indicated the possible sites of contact between Rz and T cells. The effect of serotonin, released by Rz cells, is discussed with respect to its possible physiological significance.