Changes in synaptic transmission and protein expression in the brains of adult offspring after prenatal inhibition of the kynurenine pathway.

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system.

Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are five subtypes that are all expressed in the brain of mammals (m1-m5). There are regional differences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been difficult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3H-NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo effects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents.

Muscarinic toxin selective for m4 receptors impairs memory in the rat.

The selectivity of the muscarinic toxin MT3 from green mamba snake venom was corroborated by inhibition of the binding of [3H]NMS, a classical muscarinic radioligand, to native and cloned muscarinic receptors, showing 214-fold higher affinity for m4 than for m1 subtype, without significant binding to the others. The highest concentrations of MT3 sites (putative m4 receptors) in the rat brain were found in striatum and olfactory tubercle, intermediate concentration in dentate gyrus and CA1, and lower but still conspicuous levels in CA3 and frontal cortex. MT3 caused retrograde amnesia of an inhibitory avoidance task, when injected into the dorsal hippocampus of rats after training, suggesting a positive role of these MT3 sensitive sites, which are probably m4 muscarinic receptors, in memory consolidation of this task.

Cholinergic neurotransmission and synaptic plasticity concerning memory processing.

The brain is able to change the synaptic strength in response to stimuli that leave a memory trace. Long-term potentiation (LTP) and long-term depression (LTD) are forms of activity-dependent synaptic plasticity proposed to underlie memory. The induction of LTP appears mediated by glutamate acting on AMPA and then on NMDA receptors. Cholinergic muscarinic agonists facilitate learning and memory. Acetylcholine depolarizes pyramidal neurons, reduces inhibition, upregulates NMDA channels and activates the phosphoinositide cascade. Postsynaptic Ca2+ rises and stimulates Ca-dependent PK, promoting synaptic changes. Electroencephalographic desynchronization and hippocampal theta rhythm are related to learning and memory, are inducible by cholinergic agonists and elicited by hippocampal cholinergic terminals. Their loss results in memory deficits. Hence, cholinergic pathways may act synergically with glutamatergic transmission, regulating and leading to synaptic plasticity. The stimulation that induces plasticity in vivo has not been established. The patterns for LTP/LTD induction in vitro may be due to the loss of ascending cholinergic inputs. As a rat explores pyramidal cells fire bursts that could be relevant to plasticity.

Muscarinic toxins from the venom of Dendroaspis snakes with agonist-like actions.

The venom of some Dendroaspis snakes contains small proteins (7500 mol. wt) that inhibit the binding of radiolabelled muscarinic antagonist to brain synaptomal membranes. There were no peptides described among muscarinic ligands until Adem et al. (Biochim. biophys. Acta 968, 340-345, 1988) reported that muscarinic toxins (MTxs), MTx1 and 2 were able to inhibit 3H-QNB binding to rat brain membranes. Since MTxs inhibit around half of specific binding of 3H-quinuclidinyl benzilate (3H-QNB) and 3H-N-methyl-scopolamine (3H-NMS), which do not discriminate between subtypes of muscarinic receptors, it has been proposed that MTxs might selectively bind to some subtype. MTx1 and 2 from Dendroaspis angusticeps almost completely inhibit the binding of 3H-pirenzepine (3H-PZ), a preferential M1 muscarinic receptor subtype ligand to cerebral cortex synaptosomal membranes. A much higher concentration was needed to inhibit partially 3H-PZ binding to atrial muscarinic receptors. These results support the hypothesis that MTx1 and 2 may be M1 selective muscarinic ligands. Similar activities have been found in Dendroaspis polylepis and D. viridis venoms, but with lower affinities. The Ki obtained from inhibition curves of the binding of 3H-PZ showed that MTx1 has higher affinity for the putative M1 muscarinic receptor subtype, followed by MTx2. DpMTx has lower affinity, while DvMTx seems to have the lowest affinity. All these peptides are devoid of anticholinesterase activity. Dendrotoxin and fasciculin from D. angusticeps venom do not inhibit the binding of muscarinic radioligands to cerebral cortex membranes. The injection of MTxs into dorsal hippocampus of rats immediately after training in an inhibitory avoidance task improves memory consolidation, as does oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of muscarinic toxins MTx1 and MTx2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors.

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M1 subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3H-N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for m1 receptors: Ki for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and Ki for MTx2 were 364 nM at m1 and 1.2 microM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M1-agonists McN-A-343 and CPCP (4-[N-(chlorophenyl)carbamoyloxy]-4-20-ynyl-trimethylammoniu m iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors appears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.