Giorgio Bernardi

Long-term synaptic depression in the striatum: physiological and pharmacological characterization

The effect of tetanic activation of corticostriatal glutamatergic fibers was studied in striatal slices by utilizing extracellular and intracellular recording techniques. Tetanic stimulation produced a long- term synaptic depression (LTD) (> 2 h) of both extracellularly recorded field potentials and intracellularly recorded EPSPs. LTD was not coupled with changes of intrinsic membrane properties of the recorded neurons. In some neurons, repetitive cortical activation produced a short-term posttetanic potentiation (1–3 min). Subthreshold tetanic stimulation, which under control condition did not cause LTD, induced LTD when associated with membrane depolarization. Moreover, LTD was not expressed in cells in which the conditioning tetanus was coupled with hyperpolarization of the membrane. Bath application of aminophosphonovalerate (30–50 microM), an antagonist of NMDA receptors, did not affect the amplitude of the synaptic potentials and the expression of LTD. Striatal LTD was significantly reduced by the pretreatment of the slices with 30 microM 2-amino-3-phosphonopropionic acid, an antagonist of glutamate metabotropic receptors. LTD was not blocked by bicuculline (30 microM), a GABA(A) receptor antagonist. Scopolamine (3 microM), an antagonist of muscarinic receptors, induced a slight, but significant, increase of the amplitude of LTD. Both SCH 23390 (3 microM), an antagonist of D1 dopamine (DA) receptors, and I- sulpiride (1 microM), an antagonist of D2 DA receptors, blocked LTD. LTD was also absent in slices obtained from rats in which the nigrostriatal DA system was lesioned by unilateral nigral injection of 6-hydroxydopamine. In DA-depleted slices, LTD could be restored by applying exogenous DA (30 microM) before the conditioning tetanus. In DA-depleted slices, LTD could also be restored by coadministration of SKF 38393 (3–10 microM), a D1 receptor agonist, and of LY 171555 (1–3 microM), a D2 receptor agonist. Application of a single class of DA receptor agonists failed to restore LTD. These data show that striatal LTD requires three main physiological and pharmacological conditions: (1) membrane depolarization and action potential discharge of the postsynaptic cell during the conditioning tetanus, (2) activation of glutamate metabotropic receptors, and (3) coactivation of D1 and D2 DA receptors. Striatal LTD may alter the output signals from the striatum to the other structures of the basal ganglia. This form of synaptic plasticity can influence the striatal control of motor activity.

Isochores and the evolutionary genomics of vertebrates.

The nuclear genomes of vertebrates are mosaics of isochores, very long stretches (>>300kb) of DNA that are homogeneous in base composition and are compositionally correlated with the coding sequences that they embed. Isochores can be partitioned in a small number of families that cover a range of GC levels (GC is the molar ratio of guanine+cytosine in DNA), which is narrow in cold-blooded vertebrates, but broad in warm-blooded vertebrates. This difference is essentially due to the fact that the GC-richest 10-15% of the genomes of the ancestors of mammals and birds underwent two independent compositional transitions characterized by strong increases in GC levels. The similarity of isochore patterns across mammalian orders, on the one hand, and across avian orders, on the other, indicates that these higher GC levels were then maintained, at least since the appearance of ancestors of warm-blooded vertebrates. After a brief review of our current knowledge on the organization of the vertebrate genome, evidence will be presented here in favor of the idea that the generation and maintenance of the GC-richest isochores in the genomes of warm-blooded vertebrates were due to natural selection.

Compositional constraints and genome evolution

SummaryNucleotide sequences of all genomes are subject to compositional constraints that (1) affect, to about the same extent, both coding and noncoding sequences; (2) influence not only the structure and function of the genome, but also those of transcripts and proteins; (3) are the result of environmental pressures; and (4) largely control the fixation of mutations. These findings indicate (1) that noncoding sequences are associated with biological functions; (2) that the organismal phenotype comprises two components, the classical phenotype, corresponding to the “gene products,” and a “genome phenotype,” which is defined by the compositional constraints; and (3) that natural selection plays a more important role in genome evolution than do random events.

Long‐term Potentiation in the Striatum is Unmasked by Removing the Voltage‐dependent Magnesium Block of NMDA Receptor Channels

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 μM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), an antagonist of dl‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30–50 μM 2‐amino‐5‐phosphonovalerate (APV), an antagonist of N‐methyl‐d‐aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long‐term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+‐free medium, cortically evoked EPSPs revealed an APV‐sensitive component; in this condition tetanic stimulation produced long‐term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30–50 μM APV blocked striatal LTP, while it did not affect LTD. In Mg2+‐free medium, incubation of the slices in 10 μM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long‐term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage‐dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.

Post-ischemic brain damage: pathophysiology and role of inflammatory mediators

Neuroinflammatory mediators play a crucial role in the pathophysiology of brain ischemia, exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. Within hours after the ischemic insult, increased levels of cytokines and chemokines enhance the expression of adhesion molecules on cerebral endothelial cells, facilitating the adhesion and transendothelial migration of circulating neutrophils and monocytes. These cells may accumulate in the capillaries, further impairing cerebral blood flow, or extravasate into the brain parenchyma. Infiltrating leukocytes, as well as resident brain cells, including neurons and glia, may release pro‐inflammatory mediators, such as cytokines, chemokines and oxygen/nitrogen free radicals that contribute to the evolution of tissue damage. Moreover, recent studies have highlighted the involvement of matrix metalloproteinases in the propagation and regulation of neuroinflammatory responses to ischemic brain injury. These enzymes cleave protein components of the extracellular matrix such as collagen, proteoglycan and laminin, but also process a number of cell‐surface and soluble proteins, including receptors and cytokines such as interleukin‐1β. The present work reviewed the role of neuroinflammatory mediators in the pathophysiology of ischemic brain damage and their potential exploitation as drug targets for the treatment of cerebral ischemia.

An analysis of eukaryotic genomes by density gradient centrifugation

DNA preparations from 25 eukaryotes, ranging from yeast to man, were analyzed in their base composition, sedimentation coefficient, modal and mean buoyant density in CsCl. Four mammalian and two amphibian DNAs were fractionated by preparative Cs2SO4−Ag+ density gradient centrifugation. The CsCl band profile of each fraction was analyzed in terms of Gaussian curves; this allowed the buoyant densities and the relative amounts of the components present in each DNA to be assessed. Direct analysis of the CsCl band profiles of unfractionated DNA showed a satisfactory agreement with the results obtained in the combined Cs2SO4−Ag+, CsCl investigations. This simpler procedure was therefore applied to all 25 eukaryotic DNAs. The main finding of this work is the recognition of phylogenetic differences at the macromolecular level in the organization of eukaryotic genomes. The differences concerning the main band DNA are: (a) the three major components (1·697, 1·704 and 1·709 g/cm3) first observed in the main band of calf DNA by Filipski et al. (1973) were also found in the other ten mammalian DNAs investigated here; similar components appear to be present in avian genomes; in both cases, the 1·704 and 1·709 g/cm3 components are responsible for the skewness to the heavy side of the CsCl main band of these DNAs; (b) the DNAs of reptiles, amphibians and fish showed a much lower and decreasing skewness of their CsCl bands compared to both mammalian and avian DNAs; the Cs2SO4−Ag+, CsCl combined analysis of two amphibian DNAs revealed discrete components, different from those of mammals and birds; (c) essentially symmetrical bands in CsCl were exhibited by three invertebrate DNAs; (d) the DNAs from three unicellular eukaryotes exhibited perfectly symmetrical bands in CsCl and could not be resolved into discrete comoponents. A number of observations on minor and satellite DNA components collected in this work are reported.

Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones

The interactions between N‐methyl‐d‐aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) were investigated in striatal slices, by utilizing intracellular recordings, both in current‐ and voltage‐clamp mode. Bath‐application (50 μm) or focal application of NMDA induced a transient membrane depolarization, while in the voltage‐clamp mode, NMDA (50 μm) caused a transient inward current. Following bath‐application of the non‐selective mGluR agonist 1S,3R‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD, 10 μm), NMDA responses were reversibly potentiated both in current (197±15% of control) and voltage‐clamp experiments (200±18% of control). Bath‐application of the group I mGluR agonist (RS)‐3,5‐dihydroxyphenylglycine (3,5‐DHPG, 10–300 μm) resulted in a dose‐dependent potentiation of NMDA‐induced membrane depolarization (up to 400±33% of control). This potentiation was either prevented by preincubation with (RS)‐α‐methyl‐4‐carboxyphenylglycine (RS‐α‐MCPG, 300 μm), or blocked when applied immediately after 3,5‐DHPG wash‐out. Neither (2S,1′S,2′S)2‐(2′‐carboxycyclopropyl)glycine (L‐CCG I, up to 100 μm) nor (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)‐glycine (DCG‐IV, 1 μm), agonists for group II mGluRs caused any change in NMDA responses. Likewise, l‐serine‐O‐phosphate (l‐SOP, 30 μm), agonist for group III mGluRs, did not affect the NMDA‐induced depolarization. The enhancement of the NMDA responses was mimicked by phorbol‐12,13‐diacetate (PDAc, 1 μm) which activates protein kinase C (PKC). The 3,5‐DHPG‐mediated potentiation of the NMDA‐induced depolarization was prevented by preincubation with staurosporine (100 nm) or calphostin C (1 μm), antagonists of PKC. Electrophysiological responses to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor activation were not affected by agonists for the three‐classes of mGluRs. The present data suggest that group I mGluRs exert a positive modulatory action on NMDA responses, probably through activation of PKC. This functional interaction in the striatum appears of crucial importance in the understanding of physiological and pathological events, such as synaptic plasticity and neuronal death, respectively.

PROPERTIES OF THE HYPERPOLARIZATION-ACTIVATED CATION CURRENT IH IN RAT MIDBRAIN DOPAMINERGIC NEURONS

Intracellular electrophysiological recordings in current‐ and voltage‐clamp mode were obtained from dopaminergic neurons of the rat mesencephalon in an in vitro slice preparation. In current‐clamp mode, a time‐dependent anomalous rectification (TDR) of the membrane was observed in response to hyperpolarizing current pulses. In single‐electrode voltage‐clamp mode, a slowly developing inward current (lh) underlying the TDR was studied by hyperpolarizing voltage commands from a holding potential of ‐50 to ‐60 mV. lh started to be activated at ‐69 mV, was fully activated at ‐129 to ‐141 mV, with half‐maximal activation at ‐87 mV, and showed no inactivation with time. The time course of development of Ih followed a single exponential, and its time constant was voltage‐dependent. At ‐81 mV, lh activated with a time constant of 379 2 47.6 ms, whereas at ‐129 mV lh activated with a time constant of 65 ? 2.2 ms. Its estimated reversal potential was ‐35 ± 4 mV. Raising the extracellular concentration of K+ from 2.5 to 6.5 and to 12.5 mM increased the amplitude of lh while reducing the extracellular concentration of Na+ from 153.2 to 27.2 mM caused a reduction in amplitude of lh. Bath application of caesium (1–5 mM) reversibly reduced or blocked the TDR/lh. Perfusion of tetrodotoxin (0.5–1 μM), tetraethylammonium (10–20 mM) or barium (0.3–2 mM) did not significantly affect lh. lh was also present in cells impaled with CsCI‐filled electrodes. When lh was substantially reduced by extracellular caesium (1 mM) the firing rate of the dopaminergic cells, which consisted of a spontaneous pacemaker discharge of action potentials, was not clearly changed. In addition, the holding current in voltage‐clamp experiments at ‐50 to ‐60 mV was not affected by 1 mM caesium. We conclude that although the lh current is a typical feature of the dopaminergic neurons, it is neither a significant factor underlying the spontaneous pacemaker activity nor does it contribute substantially to the setting of the normal resting potential level of the membrane. On the other hand, since lh starts at voltages lower than or equal to ‐69 mV (below firing threshold), it may play a modulatory role in the cells excitability by limiting the amplitude and duration of any prolonged hyperpolarizing events in the dopaminergic cells.

An approach to the organization of eukaryotic genomes at a macromolecular level.

DNAs from three mammals, two amphibians, two invertebrates and a unicellular eukaryote, enzymatically degraded to molecular weights in the 1×106 to 2×106 range, were analyzed in their sedimentation coefficients, and modal and mean buoyant density of their CsCl bands. These were further analyzed in terms of Gaussian curves; a more detailed analysis of these genomes was done using the combined Cs2SO4−Ag+, CsCl approach of Thiery et al. (1976). The major components of degraded mammalian DNAs were identical, in both buoyant densities and relative amounts, to those of the undergraded preparations, with only slight density shifts for some components. In contrast, the major components of degraded amphibian DNAs showed a very different pattern compared to the undergraded DNAs; changes in buoyant densities and amounts of some components, the disappearance of some components and the appearance of the components were observed. Finally, the degraded Drosophila and Saccharomyces cerevisiae DNAs were similar to the undergraded preparations in that no components could be resolved within the main bands. Mouse DNA preparations ranging in molecular weight from 1·8×106 to over 200×106 were examined. Only very slight changes in the relative amounts of some components seemed to take place in the 1·8×106 to 66×106 molecular weight range: the buoyant densities of the components did not appear to vary in the size range, 1·8×106 to over 200×106.

The gene distribution of the human genome

Linear correlations exist between the GC levels of third codon positions (GC3) of individual human genes and the GC levels of long genomic sequences and DNA molecules (50-100 kb in size) embedding the genes. These linear relationships allow the positioning of the GC3 histogram of cDNA sequences from the databases relative to the CsCl profile of human DNA. In turn, this allows an estimate of the relative concentrations of genes in genomic regions of different GC content. An estimate obtained by using current sequence data and Gaussian decompositions of the GC3 histogram and of the CsCl profile indicates that the GC-richest (non-ribosomal) component of the human genome is at least 17 times as gene-rich as the GC-poor regions. Moreover, our results suggest that the most recent physical maps of the human genome consisting of overlapping YACs cover less than 50% of the genes.