Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex

By means of a semi-automatic image analyzer plugged into an Apple II computer and suitable computer programs it is possible to analyze transmitter-identified nerve terminals. Thus, a densitometric approach is applied on the original photograph followed by systematic sampling which is carried out by means of a grating of circles. This procedure allows the study quantitatively of density and intensities of different types of densely packed tyrosine hydroxylase (TH) immunoreactive nerve terminals of the nuc. caudatus putamen. It is shown that the islandic TH immunoreactive nerve terminals have a higher density, but a TH content similar to the diffuse types of TH immunoreactive nerve terminals in the nuc. caudatus putamen.     

Perinatal methylazoxymethanol acetate uncouples coincidence of orientation of cerebellar folia and parallel fibers

Perinatal administration of methylazoxymethanol acetate in the rat, as a one time injection on gestational day 21, postnatal days 0, 1 or 2, altered the parallel orientation of cerebellar folia. The effect persisted into adulthood. In animals injected on one of the postnatal days 3, 4 or 5, the folial pattern was not altered. Even when the injection was repeated for three days on postnatal days 3, 4 and 5, changes in the cerebellar surface were not found. However, in animals receiving a low protein diet during the last five days of gestation, the three injection regimen produced a distortion of the folial pattern. The surface of cerebella of animals injected on gestational day 21 through postnatal day 2 was covered with small blebs resembling the surface of a cauliflower head. In sagittal sections, islands of cortical laminae appeared to be isolated from the arbor vitae. However, serial reconstruction of the granular layer from sections revealed that these pieces were continuous with the arbor vitae. Surprisingly, cerebella having malaligned folia also had varying degrees of Purkinje cell somas distributed throughout the granule cell layer rather than in a single layer. This occurred even when the granule cell layer approached normal thickness. Analysis of cerebellar weight from the group injected on the day of birth revealed three levels of weight reduction: severe (greater than 40%), moderate (20-40%) and mild (less than 20%). The granule cell deficit was directly related to the weight reduction of the cerebella. In the severely-affected cerebella, areas of the cortex were virtually devoid of granule cells. The moderately-affected cerebella had a continuous granular layer which was thick and thin. In the mild type, the layer was relatively normal in thickness but, nevertheless, the cerebellar surface was highly distorted. In all animals treated with methylazoxymethanol acetate on days G21 through P5, parallel fibers were disoriented. This occurred even though the folia appeared normal in the G20, P3, P4, P5 and P3-5 injected groups. Bundles of parallel fibers crisscrossed in the plane of the cerebellar surface in all areas where a molecular layer was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

The glutamate receptor subtype mediating parallel fibre-Purkinje cell transmission in rabbit cerebellar cortex

Responses of Purkinje cells to parallel fibre stimulation and to ionophoretically applied glutamate agonists, L-glutamate, L-aspartate, quisqualate, kainate and N-methyl-D-aspartate (NMDA), were extracellularly recorded in a superficial folium of the dorsal paraflocculus of high-decerebrate rabbits. NMDA caused an inhibition of simple spike discharge from Purkinje cells. 2-Amino-5-phosphonovalerate (APV), a selective NMDA receptor antagonist, effectively antagonized this inhibition. However, APV did not antagonize the excitation of Purkinje cells caused by quisqualate or parallel fibre stimulation. By contrast, both kynurenate and gamma-D-glutamylglycine (gamma-DGG) effectively antagonized the excitation of Purkinje cells by parallel fibre stimulation in a dose-dependent manner. Both kynurenate and gamma-DGG also antagonized the excitation of Purkinje cells caused by quisqualate, L-glutamate, L-aspartate or kainate. However, the antagonism was more prominent to L-aspartate and kainate than to quisqualate and L-glutamate. Quisqualate response was antagonized to an extent comparable with the response to parallel fibre stimulation. These results indicate that the receptor for the neurotransmitter at parallel fibre-Purkinje cell synapses is of quisqualate-specific type. The present data are consistent with the evidence that L-glutamate is the endogenous transmitter at these synapses.     

Clustering of the group III metabotropic glutamate receptor 4a at parallel fiber synaptic terminals in the rat cerebellar molecular layer.

We report in this study with a pre-embedding immunogold method, the clustering of the group III metabotropic glutamate receptor 4a (mGluR4a) along the presynaptic membrane of parallel fiber synaptic terminals in the cerebellar molecular layer. The mGluR4a clusters were homogeneously distributed and interspaced by about 60 nm. These results suggest a particular arrangement of mGluR4a which might help to a rapid and effective activation of this receptor by glutamate.     

The length of parallel fibers in the cat cerebellar cortex. An experimental light and electron microscopic study

Summary Experimental light and electron microscopic studies were carried out to determine the length of parallel fibers in the cat cerebellar cortex. Using a fine surgical knife, vermal and hemispheral folia were cut perpendicular to their long axis. The animals were sacrificed 1–10 days after the operation. Sections of the transected folia were then stained with a Fink-Heimer procedure. The resulting degeneration appeared as fine dots that extended lateral to the lesion, as predictable from the course of the parallel fibers. Densitometer readings indicate that the density of degeneration declines gradually lateral to the lesion. The specificity of the silver impregnation was checked by processing silver stained sections for electron microscopy. This confirmed the location of the silver precipitate on degenerating parallel fibers. The pattern of parallel fiber degeneration in the molecular layer has a trapezoidal configuration centered on the lesion. The shorter parallel fibers are located at the base of the molecular layer and extend for 5 mm. The parallel fibers become progressively longer as they approach the pial surface where they attain a maximum length of 7 mm. Our studies suggest that in folia longer than 7 mm parallel fibers are 6 mm long on the average.In addition, it was determined on Golgi sections that the average center-to-center distance between en passant boutons of individual parallel fibers is 5.2 m. The data indicate that an average parallel fiber, 6 mm long, forming approximately 1100 boutons, may synapse with each Purkinje dendritic tree it traverses.     

Parasagittal compartmentation of cerebellar mossy fibers as revealed by the patterned expression of vesicular glutamate transporters VGLUT1 and VGLUT2

The cerebellum receives sensory signals from spinocerebellar (lower limbs) and dorsal column nuclei (upper limbs) mossy fibers. In the cerebellum, mossy fibers terminate in bands that are topographically aligned with stripes of Purkinje cells. While much is known about the molecular heterogeneity of Purkinje cell stripes, little is known about whether mossy fiber compartments have distinct molecular profiles. Here, we show that the vesicular glutamate transporters VGLUT1 and VGLUT2, which mediate glutamate uptake into synaptic vesicles of excitatory neurons, are expressed in complementary bands of mossy fibers in the adult mouse cerebellum. Using a combination of immunohistochemistry and anterograde tracing, we found heavy VGLUT2 and weak VGLUT1 expression in bands of spinocerebellar mossy fibers. The adjacent bands, which are in part comprised of dorsal column nuclei mossy fibers, strongly express VGLUT1 and weakly express VGLUT2. Simultaneous injections of fluorescent tracers into the dorsal column nuclei and lower thoracic–upper lumbar spinal cord revealed that upper and lower limb sensory pathways innervate adjacent VGLUT1/VGLUT2 parasagittal bands. In summary, we demonstrate that VGLUT1 and VGLUT2 are differentially expressed by dorsal column nuclei and spinocerebellar mossy fibers, which project to complementary cerebellar bands and respect common compartmental boundaries in the adult mouse cerebellum.     

Evidence for glutamate as a neurotransmitter in the cat vestibular nerve: radioautographic and biochemical studies

Summary Evidence that glutamate acts as a neuro-transmitter in vestibular nerve fibers was sought (1) by electron microscope radioautographic identification of the uptake sites of [³H]-glutamic acid after incubation of slices of cat vestibular nuclei, and (2) by measuring changes in sodium-dependent high affinity glutamate uptake in nerve endings containing homogenates from normal and deafferented vestibular nuclei 8 to 11 days after unilateral vestibular nerve lesion. Electron microscopic radioautography revealed that glutamate had been taken up by numerous nerve endings projecting over the whole vestibular nuclear complex. The biochemical approach indicated that after section of the vestibular nerve, a significant decrease in high affinity glutamate uptake occurred in the vestibular nuclei, which lost their exclusively ipsilateral projection. This decrease varied from one area of the deafferented vestibular nuclei to another, reaching –58% in the lateral area of the central part corresponding to the ventral lateral vestibular nucleus and the rostral part of the descending vestibular nucleus. It is concluded that glutamate (or aspartate) is used by the vestibular nerve fibers as a neurotransmitter in the vestibular nuclei.     

Evidence for glutamate as a neurotransmitter in spinothalamic tract terminals in the posterior region of owl monkeys

Previous studies have suggested that glutamate is a neurotransmitter in ascending somatosensory pathways to the thalamus. The present study examined with quantitative immunohistochemical methods the presence of glutamate in spinothalamic tract terminals of owl monkeys (Aotus trivirgatus). Such terminals in the posterior region, in which a nucleus was recently identified as a specific pain and temperature relay in macaques and humans, were labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, injected into the spinal dorsal horn. Glutamate-like immunoreactivity was demonstrated with a postembedding immunogold procedure using a well-characterized glutamate antiserum. Quantitative analysis of the immunogold labeling demonstrated that the spinothalamic tract terminals contained more than twice the tissue average of glutamate-like immunoreactivity. Enrichment of glutamate-like immunoreactivity was also found in terminals of presumed cortical origin. Presynaptic dendrites, cell bodies and non-vesicle-containing dendrites diplayed low levels of glutamate-like immunoreactivity. A strong positive correlation (r=0.69; P<0.0001) was found between the density of synaptic vesicles and the density of gold particles in spinothalamic tract terminals, in contrast to a weak negative relationship (r= -0.28; P=0.089) present in GABAergic presynaptic dendrites. These data provide strong evidence that the gold labeling in the spinothalamic tract terminals represents transmitter labeling, implying that glutamate is a neurotransmitter for ascending nociceptive and thermoreceptive information in primates.     

Imaging of glutamate neurotransmitter alterations in Alzheimer's disease

Glutamate (Glu) is a major excitatory neurotransmitter in the brain and has been shown to decrease in the early stages of Alzheimer's disease (AD). Using a glutamate chemical (amine) exchange saturation transfer (GluCEST) method, we imaged the change in [Glu] in the APP‐PS1 transgenic mouse model of AD at high spatial resolution. Compared with wild‐type controls, AD mice exhibited a notable reduction in GluCEST contrast (~30%) in all areas of the brain. The change in [Glu] was further validated through ¹H MRS. A positive correlation was observed between GluCEST contrast and ¹H MRS‐measured Glu/total creatine ratio. This method potentially provides a novel noninvasive biomarker for the diagnosis of the disease in preclinical stages and enables the development of disease‐modifying therapies for AD. Copyright © 2012 John Wiley & Sons, Ltd.     

Aspartate and glutamate as synaptic transmitters of parallel visual cortical pathways

Summary Push-pull cannulae were inserted into both medial and lateral banks of the suprasylvian sulcus and used for local perfusion with artificial extracellular fluid (aECF). Electrical stimulations of regions of cortex projecting to the lateral suprasylvian area (LSA) were accompanied by enhanced levels of release of excitatory amino acids. Electrical stimulation of the area 17/18 border evoked a greater release of aspartate relative to glutamate in the medial bank of the LSA (posteromedial lateral suprasylvian: PMLS), of glutamate over aspartate in the lateral bank (posterolateral lateral suprasylvian: PLLS) while in the fundus, both were released equally or glutamate levels were slightly elevated over those of aspartate. These data support and extend the earlier proposition (Hicks and Guedes 1983) that an excitatory amino acid mediates synaptic transmission within visual cortico-cortical pathways.     

An in-silico model of the biosynthesis of neurotransmitter glutamate, elucidates the complex regulatory role of glucocorticoids in neurotransmitter glutamate release

An in-silico model, of the glucocorticoid regulated glutamate release, in rat hippocampal tissue, is constructed. The model permits the pseudo-steady state estimation of various fluxes, experimentally impossible to measure, from a set of measured rates. Estimates of the astrocytic pyruvate carboxylase reaction and the neuronal TCA cycle rates are correlated with different dexamethasone concentrations, in order to extrapolate explicit kinetic equations. The model suggests that the observed effects of glucocorticoids can be attributed to the inhibitory actions of dexamethasone on two competing pathways, that of the neuronal TCA cycle and the biosynthetic pathway of neurotransmitter glutamate.     

Biochemical evidence for glutamate as neurotransmitter in corticostriatal and corticothalamic fibres in rat brain

The effects of ablation of frontal, occipital or entire hemicortex on several neurotransmitter parameters in the rostral and caudal neostriatum, thalamus and the contralateral anterior medial cortex were investigated. In particular the effects on the high affinity uptake ofd-aspartate and on the endogenous level of amino acids, especially glutamate and aspartate, were studied in order to identify glutamate- or aspartate-containing nerve terminals in these regions.The results show a specific decrease in high affinity uptake ofd-aspartate in both rostral and caudal neostriatum ipsilateral to the lesion after frontal or entire hemidecortication. There was also a small but significant decrease ind-aspartate uptake on the contralateral side. Only the level of endogenous glutamate decreased in the neostriatum after hemidecortication. There was a specific decrease ind-aspartate uptake in the thalamus only ipsilateral to the cortical lesions. In thalamus there was a significant decrease both in the level of glutamate and to a smaller extent in that of aspartate after hemidecortication. Anterior medial cortex showed a very active high affinity uptake ofd-aspartate, which was slightly reduced after removal of the contralateral hemicortex. The high affinity uptake ofd-aspartate was in all cases mainly due to uptake in synaptosomes.The results show that the neostriatum receives glutamate-containing fibres from the neocortex, particularly the frontal part. This projection is mainly ipsilateral with a small element derived from the contralateral side. The thalamus, both the rostral and caudal parts, receives glutamate-containing fibres from the whole extent of the ipsilateral neocortex. Some of the corticothalamic fibres may also contain aspartate. The anterior medial cortex probably contains a high proportion of glutamate- and/or aspartate-containing nerve terminals but only a low proportion of these are derived from the contralateral cortex.     

Why run parallel fibers parallel? Teleostean Purkinje cells as possible coincidence detectors, in a timing device subserving spatial coding of temporal differences.

The present paper explores the possible functional significance of the parallel orientation of parallel fibers in teleostean cerebellar and cerebelloid molecular layers, taking advantage of the restricted width of these molecular layers compared with mammalian ones and several specific configurations of granule cells. These configurations include: (i) a unilateral location, i.e. at only one (lateral) side of the molecular layer, giving rise to parallel fibers without bifurcation in a unidirectional molecular layer, where all parallel fibers conduct signals in the same direction; (ii) a bilateral location at both sides of the molecular layer giving rise to a bidirectional molecular layer where parallel fibers conduct signals in two opposite directions originating from two discrete sources; and (iii) a basal (or sometimes apical) location underneath (or opposite to) the layer of Purkinje cells, giving rise to a bidirectional molecular layer where parallel fibers conduct signals in two opposite directions originating from a continuous range of sources. It is argued that molecular layers with a bilateral location of granule cells, exemplified by the mormyrid lobus transitorius, represent an optimal configuration for the analysis of small temporal differences (up to 4 ms) between inputs to the right and left granule cell mass, by means of detection of the site of coincidence of parallel fiber activity running from left to right and vice versa. Morphological aspects that probably optimize such a function include not only the parallel course and bilateral origin of parallel fibers, but also their small diameter, large number and co-extensive location, as well as the sagittal orientation and the presence of many spines of Purkinje cell dendrites and the presence of stellate and other inhibitory interneurons. The only assumption underlying the present coincidence detection hypothesis is that Purkinje cells are supposed to be maximally stimulated by parallel fiber input when all spines are activated in such a way that their excitatory postsynaptic potentials reach the axon hillock simultaneously. For molecular layers with a unilateral location of granule cells, exemplified by the teleostean torus longitudinalis-tectal marginal parallel fiber system, a similar coincidence detecting mechanism is proposed on the basis of the presence of two populations of parallel fibers with slightly different conduction velocities. Such a system might be suitable to adapt the location of coincidence peaks to topographic maps present in deeper layers of nervous tissue. Molecular layers with basally (or apically) located granule cells as encountered in the teleostean corpus cerebelli, are probably involved in the analysis of specific spatio-temporal input waves directed centripetally towards different Purkinje cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Repetitive firing of rat cerebellar parallel fibres after a single stimulation

The excitatory postsynaptic currents (EPSCs) evoked in Purkinje cells (PCs) by stimulating parallel fibres (PFs) usually show a single peak, but EPSCs with multiple peaks (polyphasic EPSCs) can be observed in slices from animals older than 15 days. The EPSCs remain polyphasic when the postsynaptic current is reduced (either by reducing the intensity of the PF stimulation or by adding AMPA receptor antagonists) and when the PC membrane potential is made positive. Thus the late peaks are not due to postsynaptic active currents generated in the imperfectly clamped PC, and must arise from repetitive action potentials in the PF. Extracellular recordings from granule cell (GC) somata showed that a single PF stimulation can elicit a doublet or a train of action potentials. Both the late action potentials recorded in the GCs and the late peaks of the polyphasic EPSCs recorded in the PCs were reduced or abolished by paired-pulse stimulation of the PF or by bath application of the GABA_A agonist muscimol. The late action potentials in the GCs were also suppressed by local application of muscimol around the cell body. We propose that after a single stimulation of a PF, the antidromic invasion of the ascending axon and the granule cell can trigger a doublet or a burst of action potentials which back-propagate into the PF (except for the first, which finds the PF still in its refractory period). The repetitive activation of the PF by a single stimulation could play a role in the induction of long-term depression.     

The δ2 'ionotropic' glutamate receptor functions as a non-ionotropic receptor to control cerebellar synaptic plasticity

The δ2 glutamate receptor (GluRδ2) belongs to the ionotropic glutamate receptor (iGluR) family and plays a crucial role in the induction of cerebellar long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Nevertheless, the mechanisms by which GluRδ2 regulates cerebellar LTD have remained elusive. Because a mutation occurring in lurcher mice causes continuous GluRδ2 channel activity that can be abolished by 1-naphtylacetylspermine (NASP), a channel blocker for Ca²⁺-permeable iGluRs, GluRδ2 is thought to function as an ion channel. Here, we introduced a mutant GluR δ 2 transgene, in which the putative channel pore was disrupted, into GluR δ 2 -null Purkinje cells using a virus vector. Surprisingly and similar to the effect of the wild-type GluR δ 2 transgene, the mutant GluR δ 2 completely rescued the abrogated LTD in GluR δ 2 -null mice. Furthermore, NASP did not block LTD induction in wild-type cerebellar slices. These results indicate that GluRδ2, a member of the iGluR family, does not serve as a channel in the regulation of LTD induction.