The Taiep Rat: A Neurological Mutant as a Model to Understand the Role of Glial Cells in Synaptic Transmission

Christian Bonansco, Marco Fuenzalida, Eduardo Couve Esteban Aliaga and Manuel Roncagliolo Centro de Neurobiología y Plasticidad del Desarrollo, Departamento de Fisiología, Facultad de Ciencias, Universidad de Valparaíso, CHILE

Financial support: Fondecyt, CONICYT-CHILE (Grant 1991004 to MR; Grant 1061074 to CB) and DIPUV-UV (Grants 08/2005 to CB, 15/2006 to EA; 46/2007 to MF; 40/207 to MR).

ABSTRACT INTRODUCTION

The taiep rat, initially described as a mutant, is now The taiep rat was described by Holmgren et al.,1 at the B. recognized for its reactive astrogliosis and the severe alterations Universidad Autónoma de Puebla (Puebla, México), as a of glutamatergic synaptic transmission in CNS. The mutant neurological mutant characterized by abnormal expresses a complex set of progressive molecular, morphological myelination and subsequent demyelination of the central and functional alterations affecting , astrocytes (CNS). The name, taiep, is an acronym for and . Here, we provide evidence that astrogliosis occurs the main neurological signs that appear progressively during in conjunction with asynchronic synaptic transmission and the first year of life of homocygote recessive mutants: mislocalization of mGluRs in taiep’s hippocampus. These tremor (first month), ataxia (3-4 months), immobility findings suggest that the mutation interferes key cellular episodes (5-6 months), audiogenic epilepsy (6-8 month) functions, such as protein trafficking, affecting the modulation and hindlimb paralysis (8 months onwards). Ta i e p is an of neurotransmitter release mediated by astrocytes. Knowledge autosomic recessive mutation and the gene responsible for of neuropathological mechanisms in taiep also provides an its phenotype has been initially located on rat chromosome opportunity to establish correlates with neurological diseases 9.2 The taiep rat first emerged as an interesting model to in human beings presenting similar taiep symptoms. study the pathogenesis of dysmyelinating diseases.

MORPHOLOGICAL FINDINGS

Alterations in myelin are probably due to a microtubular defect in oligodendrocytes, the glial cell responsible for myelin formation in CNS.3, 4 Myelinogenesis in the CNS is a complex and temporally regulated process that requires the concurrence of differentiated oligodendrocytes to synthesize large amounts of membrane to appropriately form myelin sheath around . The progressive accumulation of microtubules (MTs) physically bound to endoplasmic reticulum (ER) profiles of oligodendrocytes (Figure 1) constitutes one of the most relevant alterations observed in

Correspondence address: Christian Bonansco, Centro de Neurobiología y Plasticidad del Desarrollo, Departamento de Fisiología, Facultad de Ciencias, Universidad de Valparaíso, CHILE. 121 Phone: 56-32-2508057/59, E-mail: [email protected] and http://neurobiologiavalpo.blogspot.com/ Christian Bonansco

A B

Figure 1. Diagram showing the temporal progress of MT-ER complex formation in taiep oligodendrocytes. A-B Electron microscopy obtained from taiep oligodendrocytes at P15, with membrane profiles projected to process (A) and accumulated MTs are associated to ER profiles closely related to Golgi complex (B). the myelin mutant taiep rat.5, 6, 7 The accumulation of MT- severely affected along developmental stages of taiep rats. ER complexes, revealed by electron microscopy studies, The abnormal waves morphology, prolonged latencies and became evident from P9. As oligodendrocytes differentiate reduced amplitudes of sensory evoked potentials have a the defect projects membrane profiles through the good correlation with the dysmyelinating conditions oligodendrocyte processes. MT-ER complexes fill the observed in this mutant.10 oligodendrocyte cell blocking the intracellular transport of lipids and proteins necessary to form and mantain Developmental alterations of auditory evoked myelin7, 8 Several myelin proteins are decreased in levels potentials correlate with progressive and accumulation of PLP and MAG has been detect within demyelination of taiep CNS taiep oligodendrocytes given consistence to a trafficking defect.8 Thus, the taiep myelin mutant rat is characterized Auditory brainstem evoked responses (ABRs), used to by an early abnormal myelination, that produces a severe evaluate the nerve conduction along the auditory pathway, dysmyelination in the CNS. Defective myelin formation are characterized in rats by four major component waves (I affects the adequate maintenance of myelin, and the to IV) representing the activation of different levels, from severe loss of myelin progresses with a strong astrogliosis acoustic nerve to rostral parts of brainstem. After onset of over the first year of taiep mutant’s life.6, 9 Despite the ABRs (P12-P14), taiep rats and their nonaffected severity of taiep’s disorders, it is one of longest survival myelin heterozygous littermates that serves as controls showed a mutant. normal pattern of maturation for wave I (which correlates with an adequate myelination of peripheral portions of the ELECTROPHYSIOLOGICAL FINDINGS auditory nerve supported by Schwann cells). The remaining central waves were significantly and progressively prolonged The functional impact of the progressive deterioration of in latency and reduced in amplitude as brainstem portion myelin in taiep rats was first evidenced by of auditory pathway becomes completely demyelinated in electrophysiological studies. Central sensory pathways are mutant rats. The interpeak latency I-IV, corresponding to

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No. 2, April 2009 pp 121-126 122 The Taiep Rat: A Neurological Mutant..... the central conduction time (CCT) of the auditory control rats, reduced their latency and decreased their pathway, is considered as an index of brain maturation. sensitivity to paired pulse depression as a function of age, Normaly, from birth onwards, the CCT shows an revealing the strengthening of the synapse between sensitive exponential reduction, indicating higher velocity of nerve Ia fibers and alpha motoneurons. By contrast, the MSR of impulses through the auditory pathway, until it stabilizes. taiep rats failed to develop further from neonatal stage. Even when CCT is significantly longer in taiep at all ages, Near the end of the second postnatal week, the MSR during the first month it shows a moderate reduction latencies were still prolonged, and the MSR showed a revealing a positive but abnormal myelination process significantly stronger paired pulse depression. It seems (dysmyelination). Starting at the second month, as later unlikely that a conduction blocking could fully explain waves increases their latencies, reduce their amplitudes the persistence of MSR depression observed in taiep rats, and finally some of them disappear, the CCT begins to suggesting alterations in Ia-α motoneuron synaptic increase progressively, coinciding with a failure in the transmission.12 Excitatory postsynaptic potentials (EPSPs), maintenance of CNS myelin sheath with subsequent elicited in motoneurons by intralaminar bipolar stimulation, demyelination.10 revealed two types of taiep motoneurons, identified according to the temporal patterns of synaptic responses; Progressive deterioration of compound action (1) taiepSYN neurons, which showed no significant potential in taiep differences to control motoneurons, and (2) taiepASYN neurons, in which the initial fast glutamatergic EPSP was Optic nerve was preferred because it is a central tract, followed by a variable number of delayed, asynchronous totally myelinated by oligodendrocytes, without synapses, EPSP responses (for up to 300 ms).13 The abnormal and can be easy isolated maintaining its integrity and asynchronous transmission observed in the could functioning. We examined the ontogenic development of be a common pattern of synaptic activity disruption in other compound action potentials (CAP) throughout the first 6 regions of the taiep CNS. months of life for control and taiep rats. Control optic nerves (ON) develop CAPs characterized by three waves. Along Asynchronous synaptic transmission: a the first month, the CAPs of taiep rats showed a delayed generalized CNS phenotype in taiep rats? maturation, with lower amplitudes and longer latencies than controls; at P30, the conduction velocity has only a Using the well-characterized synapses between Schaffer third of the normal value. Later, as demyelination proceeds, collaterals (SCs) and CA1 pyramidal neurons in the conduction velocity of taiep ONs begins to decrease hippocampal slices,14, 15, 16 we found that, as in lumbospinal and CAPs undergo a gradual temporal dispersion. CAPs of motoneurons, in a high percentage of hippocampal taiep control and taiep showed differences in their neurons (47%) the initial EPSC evoked was frequently pharmacological sensitivity to TEA and 4-AP, two voltage followed by additional asynchronous synaptic currents + dependent K channel-blockers. As compared with TEA, (ASYN EPSC). 4-AP induced a significant increase of the amplitudes and a remarkable broadening of CAPs. After P20, unlike Several findings suggest a presynaptic component in this controls, the greater sensitivity to 4-AP exhibited by taiep alteration of synaptic function. The elevation of 2+ ONs correlates with the detachment and retraction of extracellular Ca induces an increase of ASYNEPSC rate paranodal loops suggesting that potassium conductances suggesting powerfully that asynchronicity is a consequence could regulate the excitability as demyelination of CNS of alterations in the presynaptic mechanisms that regulate axons progresses. It was concluded that the taiep rat, a the release of neurotransmitter.17 Consistently, the repetitive long-lived mutant, provides a useful model to study the stimulation induces an increment of ASYNEPSC and also consequences of partial demyelination and the mechanisms the single fiber stimulation was able to evoke asynchronic by which glial cells regulate the molecular organization activity (Figure 2). Taken together, this data suggest that and excitability of axonal membranes during development in taiep rat the disregulation of presynaptic Ca2+ is a critical and disease.11 determinant of asynchronic neurotransmitter release, and seems to represent a widely distributed phenotype in the Increased lability of monosynaptic glutamatergic transmission of taiep CNS. While the amount responses in taiep spinal cord involves abnormal of synaptic contact with asynchronic activity evoked by Ia-α motoneuron synaptic transmission stimulation increased with the age, the morphological and immunohystochemistry studies showed a normal During the early postnatal period, the monosynaptic reflex development of synaptic circuitry.18 These results suggest responses (MSR), evaluated in isolated spinal cord of that the asynchronous transmission may be due to a

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Figure 2: Schaffer collateral-CA1 tripartite synaptic model in control and taiep hippocampal circuits. A) Recording configuration of excitatory postsynaptic currents evoked (eEPSC) by conventional (a1) and single fiber stimulation (a2) in CA1 pyramidal neurons of control rats. B) Recording configuration of excitatory postsynaptic currents evoked (eEPSC) by conventional (b1) or single fiber stimulation (b2) in CA1 pyramidal neurons of taiep rats. A single stimulus of both several or single presynaptic terminals with asynchronic release (blue traces) evokes a variable number of asynchronic excitatory postsynaptic currents (ASYNEPSC, arrows). C) Hypothetical diagram of the signalling mediated by glutamate released by astrocytes in normal synapses (D). In taiep, the increased glial-transmission produces a disregulation of presynaptic calcium, presumably mediated by activation of mGluRs, altering the release of neurotransmitter. dysfunction in the glutamate release mechanisms which directional signaling with both pre and postsynaptic progressively increases during development and which are elements.21 Recently, it has been shown that glutamate not attributable to the existence of aberrant synaptic released from stimulated astrocytes increases temporarily contacts. These findings suggest that the mutation affecting the probability of neurotransmitter release, which is taiep rats interfered not only with the development of the mediated by activation of mGluRs on presynaptic electrical properties of neurons, but also with the modulation terminal.22 However, little is known about of likely effects of synaptic transmission. of disregulation of the astrocyte- interaction in neuropathological states. MOLECULAR FINDINGS IN GLIA- NEURON INTERACTION Astrogliosis in taiep rat

Actually, it is well accepted that glial cells plays an The severe astrogliosis reported in taiep rats,6, 9, 23 as a part important and critical role during development of neural of a complex glial syndrome, could be involved in disruption circuits modulating synaptogenesis, refinement of neural of calcium-dependent release mechanisms driving to circuits and .19, 20 A large number of asynchronous synaptic transmission. evidences have allowed recognizing astrocytes as excitable cells, capable of releasing glutamate in a Ca2+ dependent In the earliest description of taiep mutant phenotype, manner. Fluorimetric imaging and electrophysiological besides alterations in microtubular cytoskeleton of studies have led to propose a synaptic tripartite model, in oligodendrocytes, an alteration in astrocytic filaments was which the astrocyte participates actively through a bi- reported. Electron microscopy showed an excess of glial

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AB

Figure 3: GFAP immunoreactivity in CA1 stratum radiatum of hippocampus in control (A) and taiep (B) of 3 months. Astrocytes in taiep mutant are hypertrophied showing intense process outgrowth. filaments in astrocytes adjacent to altered oligodendrocyte mGluR6/mGluR7/mGluR8) are predominantly localized to process with no obvious increase in astrocyte number, a presynaptic elements.24 Interestingly, mGluR3 is associated feature that can be interpreted as a reactive astrocytosis.3 to astrocytic function since the expression in oriens and Leon-Chavez et al.9 found that astrocytosis progress in a radiatum of CA1 is ascribed to glial cells. regional and temporal pattern that closely resembles the course of demyelination. Reactive astrocytosis begins in We have characterized the expression pattern of mGluR2/ brainstem followed by cerebellum, cortex and 3, mGluR1a and mGluR5/1 in control and taiep rats. In diencephalon. Again no obvious change in numbers of control rats mGluR2/3 immunoreactivity is highly astrocytes was found but a strong age dependent increase segregated in a laminated fashion at the terminal zones of in glial fibrillary acidic protein (GFAP) immunoreactivity the mayor hippocampal pathways. In CA1 the highest was evident in several brain regions, especially in myelin density was in neuropil of stratun lacunosum moleculare, rich areas. The most important feature shown by taiep in CA3 in the neuropil of the mossy fiber terminal zone astrocytes was hypertrophy due to process outgrowth.9 This stratum lucidum and in dentate gyrus in hilus and outer kind of increase in GFAP is a common feature in one-third of the molecular layer. In taiep the laminated demyelinating and dysmyelinating diseases. However, we pattern of mGluR2/3 is completely lost showing a diffuse have found the same type of hypertrophic astrocytes in staining through the whole hippocampus (Figure 4). unmyelinated areas of the taiep brain, such as hippocampus Additionally, taiep showed a lower number of mGluR5/1 (Figure 3). immunopositive cells in pyramidal layer, interneurons in the hilus and stratum lucidum of CA3. The mGluR1 Mislocalisation of mGluRs in hippocampus of immunoreactivity pattern was similar in both taiep and taiep rats control.

Since the pivotal role of astrocytes signals delivering in These results show a strong alteration in metabotropic synaptic transmission, the astrogliosis could be involved in glutamate receptors distribution, especially mGluR2/3 in the synaptic alteration described in taiep. The astrocyte the hippocampus, which could be related whith the alterations in taiep, move us to investigate if others astrocytic synaptic alteration in the hippocampal . related molecules are misexpressed in this mutant. Gutamate This expression alteration and/or mislocalization of receptors metabotropic receptors (mGLURs) are important regulators can be underlined by a problem in gene expression or of hippocampal neurotransmission, specially mediating transport deficit in taiep. Knowing cytoskeletal alteration functional glia-neuron interaction. In the hippocampus in taiep oligodendrocytes, mislocalization of mGluR2/3 can mGluRs are expressed in a highly segregated fashion be explained by a previously unknown alteration in neuronal through the different hippocampal areas. Group I mGLURs or glial cytoskeleton mediated intracellular protein targeting (mGluR1/mGluR5) are localized to postsynaptic elements or ARN targeting, as it has been described in the while Group II (mGluR2/mGluR3) and group III (mGluR4/ oligodendrocytes.2

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Figure 4: Immunohistochemical localization of mGluR2/3 in hippocampus in control (A) and taiep (B) of one month age. The normal laminated segregation of mGluR2/3 is completely lost in taiep mutant.

CONCLUDING REMARKS AND 2 Song J, Carson JH, Barbarese E, Li FY, Duncan ID. Mol Cell PROJECTIONS Neurosci. 2003; 24(4):926-38. 3 Duncan ID, Lunn KF, Holmgren B, Urba-Holmgren R, Brignolo- Holmes L.J Neurocytol. 1992; 21(12):870-84. Like neurological symptoms of taiep, most of the described 4 Lunn KF, Clayton MK, Duncan ID.J Neurocytol. 1997; 26(5):267- abnormalities in neuronal and glial cells appear gradually 81. with development. Although it is known that the gene 5 Couve E, Cabello JF, Krsulovic J and Roncagiolo M. J Neurosci Res. 1997; 47: 573-581. responsible for this mutation is located on chromosome 9, 6 Krsulovic J, Couve E and Roncagliolo M. Biol Res. 1999; 32: their identity is unknown. While taiep was initially described 253-262 as a myelin mutant, the rat expresses a complex set of 7 Song J, O’connor LT, Yu W, Baas PW, Duncan ID. J Neurocytol. morphological and functional alterations, apparently 1999; 28(8):671-83. 8 O’Connor LT, Goetz BD, Couve E, Song J and Duncan ID. Mol without a specific cell lineage, which involves and Cellular Neurosci. 2000; 16: 396-407. oligodendrocytes, astrocytes and also neurons. The early 9 Leon Chavez BA, Guevara J, Galindo S, Luna J, Ugarte A, and progressive disturbances in taiep described here, such Villegas O, Mena R, Eguibar JR, Martinez-Fong D. Brain Res. as accumulation of microtubules, synaptic dysfunction, 2001; 4; 900(1):152-5. 10 Roncagliolo M, Benítez J, Eguibar JR. Audiol Neurootol. 2000; astrogliosis and mGluRs mislocalization, make it hard to 5(5):267-75. assume that all these disorders are merely secondary to 11 Roncagliolo M, Schlageter C, León C, Couve E, Bonansco C, oligodendrocytes disruptions. Even if we have no direct Eguibar JR. Brain Res. 2006 5; 1067(1):78-84. evidence of microtubule alteration in neurons and astrocyte 12 Fuenzalida M, Roncagliolo P, Bonansco C, Roncagliolo M. Brain Res Dev Brain Res. 2004; 25;153(2):197-202. we think that mechanism of microtubule assembly/ 13 Bonansco C, Fuenzalida M, Roncagliolo M. Exp Brain Res. disassembly could be altered in various cell types. So we 2004; 156(1):104-10. envisage that taiep gene is involved in cytoesqueletal 14 Bonansco C, González de la Vega A, González Alegre P, Borde structure and function that underline a key cellular M, Garcá-Segura LM, Buño W. Hippocampus. 2002;12(4):434- 46. function, such as mRNA or protein targeting and 15 Bonansco C, Buño W. Hippocampus. 2003;13(1):150-63. trafficking. 16 Cerpa W, Godoy JA, Alfaro I, Farías GG, Metcalfe MJ, Fuentealba R, Bonansco C, Inestrosa NC. J Biol Chem. 2008 The knowledge of pathological mechanisms in taiep, 29;283(9):5918-27. 17 Bonansco C, Fuenzalida M, Olivares V, Molina C, Roncagliolo which can be obtained through the study of their M. J Neurosci Res. 2007; 85(1):223-9. functional alterations, provides an opportunity to establish 18 Bonansco C, Fuenzalida M, Molina C, Olivares V and correlates with neurological or neurodegenerative diseases Roncagliolo M. Synaptic Function and Plasticity Conference, in human beings presenting some of the taiep symptoms. 2005, Vancouver, CANADA. 19 Haydon PG. Nat Rev Neurosci. 2001, 2(3):185-193. Functional studies about mechanism of asynchronic 20 Stevens B. Neurosignals. 2008, 18;16(4):278-288. synaptic transmission and their relationships with altered 21 Araque A, Parpura V, Sanzgiri RP, Haydon PG. Trends Neurosci. astroglial modulation represent a useful tool to understand 1999, 22(5):208-15. the cellular basis of epilepsy and motor control diseases. 22 Perea G, Araque A. Science. 2007, 24;317(5841):1083-6. 23 León-Chávez BA, Antonio Gonzalez-Barrios J, Ugarte A, Meraz MA, Martinez-Fong D. Brain Res. 2003, 7;965(1-2):274-8. REFERENCES 24 Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki A, Abe T, Nakanishi S, Mizuno N. J 1 Holmgren B, Urbá-Holmgren R, Riboni L, Vega-Saenz de Miera Neurosci. 1997, 1;17(19):7503-22. EC. Lab Anim Sci. 1989; 39(3):226-8.

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