Nicola Berretta

Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory

Induction of long‐term depression (LTD) in rat striatal slices revealed that this form of synaptic plasticity is coupled to an increased expression of tissue‐plasminogen activator (t‐PA) mRNA, as detected by the mRNA differential display technique. To further investigate the involvement of this gene in synaptic remodelling following striatal LTD, we recorded electrical activity from mice lacking the gene encoding t‐PA (t‐PA‐KO) and from wild‐type (WT) mice. Tetanic stimulation induced LTD in the large majority of striatal neurons recorded from WT mice. Conversely, LTD was absent in a significant proportion of striatal neurons obtained from mice lacking t‐PA. Electrophysiological recordings obtained from hippocampal slices in the CA1 area showed that mainly the late phase of long‐term potentiation (LTP) was reduced in t‐PA‐KO mice. Learning and memory‐related behavioural abnormalities were also found in these transgenic mice. Disruption of the t‐PA gene, in fact, altered both the context conditioning test, a hippocampus‐related behavioural task, and the two‐way active avoidance, a striatum‐dependent task. In an open field object exploration task, t‐PA‐KO mice expressed deficits in habituation and reactivity to spatial change that are consistent with an altered hippocampal function. Nevertheless, decreased rearing and poor initial object exploration were also observed, further suggesting an altered striatal function. These data indicate that t‐PA plays a critical role in the formation of various forms of synaptic plasticity and memory.

Inflammation Subverts Hippocampal Synaptic Plasticity in Experimental Multiple Sclerosis

Abnormal use-dependent synaptic plasticity is universally accepted as the main physiological correlate of memory deficits in neurodegenerative disorders. It is unclear whether synaptic plasticity deficits take place during neuroinflammatory diseases, such as multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). In EAE mice, we found significant alterations of synaptic plasticity rules in the hippocampus. When compared to control mice, in fact, hippocampal long-term potentiation (LTP) induction was favored over long-term depression (LTD) in EAE, as shown by a significant rightward shift in the frequency–synaptic response function. Notably, LTP induction was also enhanced in hippocampal slices from control mice following interleukin-1β (IL-1β) perfusion, and both EAE and IL-1β inhibited GABAergic spontaneous inhibitory postsynaptic currents (sIPSC) without affecting glutamatergic transmission and AMPA/NMDA ratio. EAE was also associated with selective loss of GABAergic interneurons and with reduced gamma-frequency oscillations in the CA1 region of the hippocampus. Finally, we provided evidence that microglial activation in the EAE hippocampus was associated with IL-1β expression, and hippocampal slices from control mice incubated with activated microglia displayed alterations of GABAergic transmission similar to those seen in EAE brains, through a mechanism dependent on enhanced IL-1β signaling. These data may yield novel insights into the basis of cognitive deficits in EAE and possibly of MS.

Muscarinic receptors depress gabaergic synaptic transmission in rat midbrain dopamine neurons

The effects of muscarine and nicotine on evoked and spontaneous release of GABA were studied using intracellular and whole-cell patch-clamp recordings from rat midbrain dopamine neurons in an in vitro slice preparation. Muscarine (30 microM) reversibly depressed the pharmacologically isolated inhibitory postsynaptic potential evoked by local electrical stimulation. The maximal inhibition of the inhibitory postsynaptic potential amplitude was 39.6+/-5%. This depressant effect of muscarine was blocked by the M3/M1 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (100 nM), but was slightly affected by the M1/M3 receptor antagonist pirenzepine (1 microM). In addition, muscarine decreased the frequency of the miniature synaptic currents without any effect on their amplitude. Moreover, muscarine did not change the GABA-induced hyperpolarization, indicating that its effect on the inhibitory postsynaptic potential is mediated by presynaptic receptors. On the contrary, the cholinergic agonist nicotine did not change the frequency or the amplitude of the spontaneous glutamatergic and GABAergic synaptic currents. Our data indicate that a prevalent activation of presynaptic M3 muscarinic receptors inhibits the GABA-mediated synaptic events, while the activation of nicotinic receptors does not affect the release of glutamate and GABA on midbrain dopamine neurons.

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.

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.

Interleukin-1β Promotes Long-Term Potentiation in Patients with Multiple Sclerosis

The immune system shapes synaptic transmission and plasticity in experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (MS). These synaptic adaptations are believed to drive recovery of function after brain lesions, and also learning and memory deficits and excitotoxic neurodegeneration; whether inflammation influences synaptic plasticity in MS patients is less clear. In a cohort of 59 patients with MS, we found that continuous theta-burst transcranial magnetic stimulation did not induce the expected long-term depression (LTD)-like synaptic phenomenon, but caused persisting enhancement of brain cortical excitability. The amplitude of this long-term potentiation (LTP)-like synaptic phenomenon correlated with the concentration of the pro-inflammatory cytokine interleukin-1β (IL-1β) in the cerebrospinal fluid. In MS and EAE, the brain and spinal cord are typically enriched of CD3+ T lymphocyte infiltrates, which are, along with activated microglia and astroglia, a major cause of inflammation. Here, we found a correlation between the presence of infiltrating T lymphocytes in the hippocampus of EAE mice and synaptic plasticity alterations. We observed that T lymphocytes from EAE, but not from control mice, release IL-1β and promote LTP appearance over LTD, thereby mimicking the facilitated LTP induction observed in the cortex of MS patients. EAE-specific T lymphocytes were able to suppress GABAergic transmission in an IL-1β-dependent manner, providing a possible synaptic mechanism able to lower the threshold of LTP induction in MS brains. Moreover, in vivo blockade of IL-1β signaling resulted in inflammation and synaptopathy recovery in EAE hippocampus. These data provide novel insights into the pathophysiology of MS.

Alpha1-adrenoceptor-mediated excitation of substantia nigra pars reticulata neurons

The effect of noradrenaline was studied in principal neurons of the substantia nigra pars reticulata in rat brain slices using patch clamp recordings. Perfusion of noradrenaline or the alpha(1)-adrenoceptor agonist phenylephrine increased the spontaneous firing activity of reticulata cells. The alpha(1)-adrenoceptor antagonist prazosin counteracted the effects of noradrenaline. In contrast, the beta-adrenoceptor agonist isoproterenol did not affect the activity of reticulata cells and the beta-adrenoceptor antagonist pindolol did not prevent noradrenalines effect. In whole-cell recordings, at -60 mV holding potential, noradrenaline caused a tetrodotoxin-resistant inward current with a time-course similar to the increase in firing activity. Analysis of the reversal potential of this current did not give homogeneous results. The net noradrenaline current could be associated with a conductance decrease or increase, or in some cases it did not reverse over a range from -120 to -30 mV. It is suggested that noradrenaline increases the excitability of substantia nigra reticulata cells through alpha(1)-adrenoceptors. Both a reduction and an increase in membrane conductance may mediate this effect. The increase in the tonic firing of principal reticulata cells caused by noradrenaline may have significant consequences in regulating the final output of the basal ganglia and consequently in motor-related behaviours.

Altered long-term corticostriatal synaptic plasticity in transgenic mice overexpressing human CU/ZN superoxide dismutase (GLY93→ALA) mutation

Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.

Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer’s disease

Alterations of the dopaminergic (DAergic) system are frequently reported in Alzheimers disease (AD) patients and are commonly linked to cognitive and non-cognitive symptoms. However, the cause of DAergic system dysfunction in AD remains to be elucidated. We investigated alterations of the midbrain DAergic system in the Tg2576 mouse model of AD, overexpressing a mutated human amyloid precursor protein (APPswe). Here, we found an age-dependent DAergic neuron loss in the ventral tegmental area (VTA) at pre-plaque stages, although substantia nigra pars compacta (SNpc) DAergic neurons were intact. The selective VTA DAergic neuron degeneration results in lower DA outflow in the hippocampus and nucleus accumbens (NAc) shell. The progression of DAergic cell death correlates with impairments in CA1 synaptic plasticity, memory performance and food reward processing. We conclude that in this mouse model of AD, degeneration of VTA DAergic neurons at pre-plaque stages contributes to memory deficits and dysfunction of reward processing.

Molecular and synaptic changes in the hippocampus underlying superior spatial abilities in pre-symptomatic G93A+/+ mice overexpressing the human Cu/Zn superoxide dismutase (Gly93 → ALA) mutation

Although amyotrophic lateral sclerosis (ALS) is mainly considered as a motor disease, extramotor neural and cognitive alterations have also been reported in ALS patients. There is evidence that mutations in the Cu/Zn superoxide dismutase (SOD1) gene are implicated in about 20% of familiar ALS and transgenic mice overexpressing the human Cu/Zn superoxide dismutase (GLY(93) --> ALA) mutation show an ALS-like phenotype. However, while motor behavior has been extensively analyzed in these mutants, little is known on their cognitive abilities. To characterize the pre-symptomatic cognitive profile of G93A+/+ mice, we estimated their capability to detect spatial novelty and examined several indexes of their hippocampal function. We found an enhancement of spatial abilities in mutant mice associated with (1) a higher expression of hippocampal AMPA subunit GluR1 mRNA and of GluR1 protein levels, and (2) an increased induction and maintenance of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Thus, before leading to extensive neuronal excitotoxicity, the high endogenous levels of glutamate present in the brain of pre-symptomatic G93A+/+ mice could mediate site-specific molecular and synaptic changes providing favorable conditions to spatial information processing. These findings suggest that identification of pre-symptomatic behavioral changes in murine models of ALS may point to early neural abnormalities selectively associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene.