Fabio Benfenati

Intramembrane Interactions between Neurotensin Receptors and Dopamine D2 Receptors as a Major Mechanism for the Neuroleptic‐like Action of Neurotensina

Evidence has been presented that behavioral actions of NT, inducing its neuroleptic-like action, can be explained on the basis of NT-D2 intramembrane receptor-receptor interactions in the basal ganglia, unrelated to the coexistence phenomenon, leading to reduced affinity and transduction of the D2 agonist binding site. By reducing selectively D2 receptor transduction at the pre- and postsynaptic level, the NT receptor appears capable of switching the DA synapses towards a D1 receptor-mediated transduction, illustrating how receptor-receptor interactions can increase the functional plasticity of central synapses (FIG. 12).

Chapter 19 Aspects on the information handling by the central nervous system: focus on cotransmission in the aged rat brain

Publisher Summary This chapter discusses a study on a particular animal model—the aged brain of the rat—to evaluate the possible functional meaning of cotransmission. Aging is a progressive change in the morphological and biochemical features of the organism that takes place toward the end of adult life. Regional data on the effects of aging on two basic parameters of neural function—namely, Na + /K + ATPase, which is involved in the maintenance of membrane polarization, and protein phosphorylation, which is a biochemical system related to the intracellular decoding of extracellular signals—is reported. Cyclic adenosine monophosphate and Ca 2+ -induced protein phosphorylation was carried out. The studies were performed on crude mitochondrial membrane preparations obtained from dorsal cortex, striatum, limbic system, hippocampal formation, and spinal cord. It has been shown that the density of [ 3 H]ouabain binding sites shows a gradient with the highest density in the phylogenetically oldest regions of the central nervous system (CNS) and the lowest density in the most recent ones (cerebral cortex has the lowest value), while protein phosphorylation tends to have an opposite gradient.

On the Role of Neuropeptide Y in Information Handling in the Central Nervous System in Normal and Physiopathological States

The NPY neurons play an important role in information handling in the CNS by their ability to interact in both wiring and volume transmission at the network, local circuit and synaptic level. The importance of NPY/alpha 2 receptor-receptor interactions in cardiovascular, neuroendocrine and vigilance control is emphasized. Alterations in these receptor-receptor interactions take place in the spontaneously hypertensive rats as well as in the ischemic brain, which may have profound consequences for the information handling and contribute to the functional alterations found in these pathophysiological states. Finally, in the aging brain there appears to exist a marked reduction in NPY transmission line, which may affect higher brain functions, such as learning and memory retrieval. The most impressive result is, however, the indications of a role for NPY in volume transmission, where NPY appears to produce syndromic actions via its conversion into biologically active fragments, which may have preferential actions at Y2 NPY receptors. These syndromic pathways may be altered in the spontaneously hypertensive rat and may be controlled by gonadal steroids and glucocorticoids. Glucocorticoid receptors have been demonstrated in all arcuate NPY neurons and all NA/NPY and A/NPY costoring neurons.

Modulation of Dopamine D1 and D2 Transmission Lines in the Central Nervous System

There is now general agreement about the existence of at least two distinct subtypes of dopamine (DA) receptors, the Dl and the D2 receptors (see Kebabian and Calne, 1979; Spano etai, 1979; Seeman, 1980, 1987). The Dl receptor is positively coupled to adenylate cyclase (Kebabian et al., 1972) and the D2 receptor is negatively or not coupled to adenylate cyclase (Kebabian and Calne, 1979; Stoof and Kebabian, 1984). Both the Dl and D2 receptors exist in a high-affinity and low-affinity form, the interconversion between the two states being controlled by guanyl nucleotides (see Seeman, 1977; Clark and White, 1987; Leff and Creese, 1983). The classification of Dl and D2 receptors was introduced by Kebabian and Calne (1979) (see also Spano et al., 1979) based, inter alia, on work on DAergic ergot derivatives demonstrating that many of these DA agonists had very weak ability—if any—to increase adenylate cyclase activity (Fuxe et al., 1978a, b; Fuxe, 1979; Fuxe and Calne, 1979).

Morphometrical evidence for a complex organization of tyrosine hydroxylase-,enkephalin- and darpp-32-like immunoreactive patches and their codistribution at three rostrocaudal levels in the rat neostriatum

Tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein (Mr = 32,000)-like and enkephalin-like immunoreactive profiles and their codistribution have been evaluated at three rostrocaudal levels of the rat neostriatum by means of a computer-assisted morphometrical method, which allows an objective definition of high density/intensity patches using specific antibodies in combination with the peroxidase-antiperoxidase technique. Our results show that both tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein-like and enkephalin-like profiles are organized in patches in the rat neostriatum. In the marginal zone, the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches both occupied a large part of the total area. Moreover, in this zone, these putative markers for pre- and postsynaptic elements of dopaminergic synapses also showed a complete spatial overlap. In contrast, the enkephalin-like immunoreactive patches in the marginal zone occupied a smaller area, and showed only an incomplete, albeit significant overlap with the tyrosine hydroxylase-like immunoreactive/dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive system. In the central zone, tyrosine hydroxylase-like immunoreactive, dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive and enkephalin-like immunoreactive patches occupied a much smaller part of the total area than did those in the marginal zone. Within the central zone, enkephalin-like immunoreactive patches occupied a significantly larger area than did the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches. No consistent pattern of overlap between the three different staining patterns could be seen in the central zone, probably due to the small, inconsistent size of the patches. Trend analysis showed a consistent trend of more tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the dorsal than in the ventral striatum, and a trend of more enkephalin-like immunoreactive patches in the rostral than in the caudal striatum. Our data thus demonstrate that, by using computer-assisted morphometrical techniques, it is possible to describe a non-homogenous but overlapping distribution of tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the rat neostriatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular injection of synapsin I induces neurotransmitter release in C1 neurons of Helix pomatia contacting a wrong target.

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I. These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.

A Novel Synaptic Vesicle-Associated Phosphoprotein: SVAPP-120

Generation of antibodies and direct protein sequencing were used to identify and characterize proteins associated with highly purified synaptic vesicles from rat brain. A protein doublet of low abundance of 119 and 124 kDa apparent molecular mass [synaptic vesicle‐associated phosphoprotein with a molecular mass of 120 kDa (SVAPP‐120)] was identified using polyclonal antibodies. SVAPP‐120 was found to copurify with synaptic vesicles and to be enriched in the purified synaptic vesicle fraction to the same extent as synapsin I. Like synapsin I, SVAPP‐120 is not an integral membrane protein because it was released from synaptic vesicles by high salt concentrations. This protein was demonstrated to be brain specific, and its distribution in various brain regions paralleled the distribution of synapsin I and synaptophysin. During the postnatal development of the rat cortex and cerebellum, its expression correlated with synaptogenesis. SVAPP‐120 was demonstrated to be a phosphoprotein both in vivo and in vitro. It was shown to be phosphorylated on serine and to a lesser extent on threonine residues. These results provide evidence that SVAPP‐120 represents a novel synaptic vesicle‐associated phosphoprotein. In addition, aldolase, a glycolytic enzyme, and αc‐adaptin, a clathrin assembly‐promoting protein, were identified on purified synaptic vesicles by direct protein sequencing.

Effects of chronic treatment with uridine on striatal dopamine release and dopamine related behaviours in the absence or the presence of chronic treatment with haloperidol

Uridine (15mg/kg/day, i.p.), haloperidol (1mg/kg/day, i.p.), uridine (15mg/kg/day, i.p.) plus haloperidol (1mg/kg/day, i.p.) or saline have been chronically administered to Sprague-Dawley male rats. Following 1 week of wash-out, the effects of these treatments on basal striatal dopamine (DA) release as well as on the DA release induced by an acute haloperidol challenge (2mg/kg, i.p.) were studied by means of intracerebral microdialysis. Behavioural tests such as haloperidol-induced catalepsy or apomorphine-induced stereotypics were also performed 4-7 days after drug withdrawal. The chronic treatment with uridine alone or associated with haloperidol markedly reduced DA release induced by an acute haloperidol challenge. The behavioural studies also indicated a change in DA-related behaviours in these conditions. The animals chronically treated with uridine showed significant increases in the stereotypy scores and in the catalepsy induced by an acute haloperidol challenge with respect to saline treated rats. The present results indicate that a chronic uridine treatment is able to potentiate the reduction of the striatal DA transmission induced by acute and chronic haloperidol treatment. This finding suggests the possibility to reduce the neuroleptic dose in the treatment of schizophrenia by combining neuroleptic and uridine treatments, thus making it possible to relieve some of the side effects of neuroleptic therapy.

New Evidence for the Morphofunctional Recovery of Striatal Function by Ganglioside GM1 Treatment Following a Partial Hemitransection of Rats. Studies on Dopamine Neurons and Protein Phosphorylation

In previous studies it has been shown that chronic treatment with GM1 can increase the survival of dopamine (DA) cell bodies with their dendrites in substantia nigra following a partial unilateral hemitransection of rats (Agnati et al., 1983a; 1984; Fuxe and Agnati, 1984; Toffano et al., 1983; 1984a). This in turn may be responsible for the enhancement of collateral sprouting observed from remaining striatal DA nerve terminals, leading to recovery of dopaminergic synaptic function in the striatum. This action has been found to be specific and not related to antiinflammatory effects of ganglioside GM1, since neither treatment with dexamethazone nor treatment with acetylsalicylic acid has been able to produce any trophic action on the nigral DA nerve cell bodies following a partial hemitransection in rats (Agnati et al., 1984). Recently we have also analyzed the effects of ganglioside GM1 treatment in unilateral partially hemitransected rats on striatal energy metabolism using the radioactive deoxyglycose technique and on striatal blood flow using radiolabelled iodoantipyrine as tracer (Agnati et al., 1985a). The results showed that GM1 can counteract the imbalance in striatal energy metabolism and in striatal blood flow found between the striatum of the lesioned and unlesioned side. This action may be related to excitatory effects of GM1 on the lesioned side and to inhibitory effects of GM1 on the unlesioned side. These results underline the evidence obtained in previous functional studies indicating that the metabolism of a striatum undergoing degenerative and regenerative changes can be at least partially restored following chronic GM1 treatment (Agnati et al., 1985a).

The Synapsins and the Control of Neuroexocytosis

The synapsins have been the first synaptic vesicle-associated proteins to be discovered thanks to their prominent ability to be phosphorylated by a variety of protein kinases. At present, the synapsin family in mammals consists of at least 10 isoforms encoded by three distinct genes and composed by a mosaic of conserved and variable domains. The synapsins are highly conserved evolutionarily and synapsin homologues have been described in invertebrates and lower vertebrates. The synapsins are implicated in multiple interactions with synaptic vesicle proteins and phospholipids, actin and protein kinases. Via these interactions, the synapsins play multiple roles in synaptic transmission, including control of synapse formation, regulation of synaptic vesicle trafficking, neurotransmitter release and expression of short-term synaptic plasticity phenomena. This chapter tries to summarize the main functional features of the synapsins that have emerged in the last 20 years, in order to provide a framework for interpreting the complex role played by these phosphoproteins in synaptic physiology.