Biochemical analysis of muscarinic- and metabotropic-glutamate receptor stimulated phosphoinositide (PI) turnover in rat cortical preparations during the first three weeks of postnatal development indicates the existence of a transiently increased accumulation of labeled inositol polyphosphates during the first postnatal week (Gonzales and Crews, 1985; Dudek et al., 1989). We now report for first time the visualization of those neurons responding with increased PI turnover to glutamatergic or cholinergic-receptor stimulation in rat somatosensory cortex during early postnatal development utilizing a recently described method (Bevilacqua et al, 1994). Three, 7, 10, 14, and 21 d old rats were studied. Carbachol in the presence of lithium stimulates 3H-CMP-PA accumulation throughout the cortex at all ages studied. In comparison labeled neurons responding to t-ACPD in the presence of lithium were located exclusively in layer V at P3 and P7, but were found labeled throughout the cortex at P10. Given that glutamate and cholinergic agonist stimulation are both necessary but not sufficient for cortical plasticity to occur, and that muscarinic and mGluRs stimulation both induce a peak in PI turnover response during the same period of experience-dependent neocortical plasticity, PI derived second messengers signals might be involved in the regulation of the molecular mechanisms of neuronal plasticity. Furthermore, our results show the anatomical correlate of receptor-specific PI turnover activation, and indicate that specific agonist induced PI responses are age, and layer specific.

Phosphatidylinositol bisphosphate (PtdIns(4,5)P2) hydrolysis by receptor stimulated phospholipase C (PLC) generates the ubiquitous second messengers, inositol msphosphate and diacylglycerol. The components of this signalling pathway are especially prominent in the mammalian central nervous system [1,2], but available assays of this response provide no information on the anatomical location of the responding cells, nor do they distinguish between, for example, responding neurons or glial cells. Both products of PLC activity are actively recycled to replenish inositol phospholipid pools and previous work has established that, under appropriate circumstances, CMP-phosphatidate (CMP-PA; the specific lipid precursor of inositol phospholipids), is a convenient and sensitive marker of the membranes supporting agonist-stimulated inositol lipid hydrolysis. Specifically, CMP-PA accumulates in stimulated cells provided that lithium is present to prevent resynthesis of inositol from inositol phosphates [3,4]. In the absence of lithium, inositol combines with CMP-PA to resynthesise phosphatidly inosi tol.CMP-PA is the only lipid which becomes labelled when cells are incubated with [3H]-cytidine [3,4]. Thus the accumulation of [3H]-CMP-PA was recently exploited to map the distribution of agonist stimulated inositol lipid hydrolysis in rat brain slice preparations using standard autoradiographic procedures [5]. We now report substantial modifications to the published procedures which have allowed us to visualise [3H]-CMP-PA accumulation in single neurones; combining biochemical and autoradiographic methods we demonstrate a muscarinic cholinagic receptor-stimulated increase in [3H]-CMP-PA in single hippocampal pyramidal neurones.Hippocampi from adult male Wistar rats (200-2508) were dissected on ice and chopped laterally using a McIlwain tissue chopper. The slices were recuperated in Krebs Ringer bicarbonate (KRB) for 60 min at 37OC with replenishment of fresh oxygenated KRB every 15 min. The slices were then t r a n s f e d to 2Oml flat bottomed glass bottles containing 5pCi of [3H]-cytidine in 590p1 fresh KRB and constantly flushed with 02/C02 (95/5). The slices were incubated for 30 min. before the addition of lop1 of LiCl (final concentration, 10mM) or LiCl plus carbachol (10mM and lmM, respectively) from stock solutions.Incubations were terminated after 60 min. by the addition of ice cold fixative; 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M phosphate buffered saline (PBS), pH 7.4. Cryoprotectant (20% sucrose, 8% glycerol in 0.1M PBS) was added after 45 min. and the samples were frozen in liquid nitrogen and thawed at room temperature. Ribonuclease treatment was carried out in 0.005% saponin, 3% polyethyleneglycol, 2mM EDTA, 50mM Tris-HC1, pH 7.4 for 60 min. at 3 P C and terminated by the addition of ice cold buffer. The slices were then postfixed in l%OsO4 in 0.1M PBS, dehydrated in increasing concentrations of ethanol with a final treatment in propylenoxide, infiltrated overnight and embedded in Ducurpan resin. Semit...

Dynamin-2 is a large GTP-ase, member of the dynamin superfamily, that regulates membrane remodeling and cytoskeleton dynamics. In the mammalian nervous system dynamin-2 modulates synaptic vesicle (SV)-recycling at the nerve terminals and receptor-trafficking to and from postsynaptic densities (PSDs). Mutations in dynamin-2 cause autosomal dominant centronuclear myopathy (CNM), a congenital neuromuscular disorder characterized by progressive weakness and atrophy of distal skeletal muscles. Cognitive defects have also been reported in dynamin-2-linked CNM patients suggesting a concomitant impairment of the central nervous system. Here we addressed the mechanisms that lead to cognitive defects in dynamin-2-linked CNM using a knock-in mouse model that harbors the p.R465W mutation in dynamin-2, the most common causing CNM. Our results show that these mice exhibit reduced capability to learn and acquire spatial and recognition memory, impaired long-term potentiation of the excitatory synaptic strength and perturbed dendritic spine morphology, which seem to be associated with actin defects. Together, these data reveal for the first time that structural and functional synaptic defects underlie cognitive defects in the CNM context. In addition our results contribute to the still scarce knowledge about the importance of dynamin-2 at central synapses.

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IntroductionInfarction of the anterior spinal artery territory manifests with bilateral segmental lower motor neurone weakness and, below the level of injury, pyramidal signs and loss of pain and thermal sensation. Two patients presented with neuropathic upper limb pain and flaccid paralysis ipsilateral to a unilateral infarction, no pyramidal signs and contralateral loss of thermal and pain sensation. We consider the site of vascular pathology and the mechanism of the ipsilateral pain.Methods and ResultsTwo women, 22 and 24 year-old, had acute unilateral neuropathic pain and weakness of the right upper limb and contralateral thermoalgesic sensory loss. Denervation on the side of paralysis followed. CSF and SSEPs were normal. Thermal Sensory Analysis showed thermal hypoesthesia and hypoalgesia contralateral to the pain and motor deficit. Contact heat evoked potentials were absent or smaller from the arm contralateral to the paralysis. MRIs showed abnormal signal in right anterior spinal artery territory at C1–2 to C4–5, and predominantly unilateral at C3–4 to C4–5 respectively.DiscussionThe territory matches the sulcal commisural artery (SCA), a branch of the anterior spinal artery (ASA).1 Double ASA and double SCA are described.2 This territory can also be affected by proximal pathology of the ASA, feeding radicular arteries3 and vertebral arteries.4 The ipsilateral pain may be attributed to ischaemia of ventral root afferent fibres; in the cat they supply cutaneous or visceral structures from the limbs or innervate the ventral root proper, or its sheath, and respond best to noxious stimuli.5,6 In humans the failure of dorsal rhizotomy to relieve neuropathic pain was attributed to ventral root unmyelinated axons,720% of ventral roots axons are unmyelinated8 and electrical stimulation of leg sensory nerves elicited small potentials in the ventral roots.9 ConclusionUnilateral anterior cervical cord infarction can present with segmental acute ipsilateral neuropathic pain and lower motorneuron weakness, contralateral spinothalamic loss and no pyramidal signs below the infarction level. The territory affected may be that of a sulcal commissural artery. The ipsilateral pain suggests that a subset of human afferent ventral root sensory fibres plays a functional role in pain.FundingFondecyt (Chile) Grant N°1120339.ReferencesWeidaueret al. 2015.Thorn AK. Vascular Anatomy of the spinal cord.Cheshireet al. 1996.Liet al. 2010.Cliftonet al. 1976.Janiget al. 1991.Coggeshallet al. 1975.Koet al. 2009.Phillipset al. 2000.