Fengyi Liang

Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

Silent information regulator-2 (SIR2) proteins regulate lifespan of diverse organisms, but their distribution and roles in the CNS remain unclear. Here, we show that sirtuin 2 (SIRT2), a mammalian SIR2 homolog, is an oligodendroglial cytoplasmic protein and localized to the outer and juxtanodal loops in the myelin sheath. Among cytoplasmic proteins of OLN-93 oligodendrocytes, α-tubulin was the main substrate of SIRT2 deacetylase. In cultured primary oligodendrocyte precursors (OLPs), SIRT2 emergence accompanied elevated α-tubulin acetylation and OLP differentiation into the prematurity stage. Small interfering RNA knockdown of SIRT2 increased the α-tubulin acetylation, myelin basic protein expression, and cell arbor complexity of OLPs. SIRT2 overexpression had the opposite effects, and counteracted the cell arborization-promoting effect of overexpressed juxtanodin. SIRT2 mutation concomitantly reduced its deacetylase activity and its impeding effect on OLP arborization. These results demonstrated a counterbalancing role of SIRT2 against a facilitatory effect of tubulin acetylation on oligodendroglial differentiation. Selective SIRT2 availability to oligodendroglia may have important implications for myelinogenesis, myelin–axon interaction, and brain aging.

Comparison of the Connectional Properties of the Two Forelimb Areas of the Rat Sensorimotor Cortex: Support for the Presence of a Premotor or Supplementary Motor Cortical Area

The existence of multiple motor cortical areas that differ in some of their properties is well known in primates, but is less clear in the rat. The present study addressed this question from the point of view of connectional properties by comparing the afferent and efferent projections of the caudal forelimb area (CFA), considered to be the equivalent of the forelimb area of the primary motor cortex (MI), and a second forelimb motor representation, the rostral forelimb area (RFA). As a result of various tracing experiments (including double labeling), it was observed that CFA and RFA had reciprocal corticocortical connections characterized by preferential, asymmetrical, laminar distribution, indicating that RFA may occupy a different hierarchical level than CFA, according to criteria previously discussed in the visual cortex of primates. Furthermore, it was found that RFA, but not CFA, exhibited dense reciprocal connections with the insular cortex. With respect to their efferent projection to the basal ganglia, it was observed that CFA projected very densely to the lateral portion of the ipsilateral caudate putamen, whereas the contralateral projection was sparse and more restricted. The ipsilateral projection originating from RFA was slightly less dense than that from CFA, but it covered a larger portion of the caudate putamen (in the medial direction); the contralateral projection from RFA to the caudate putamen was of the same density and extent as the ipsilateral projection. The reciprocal thalamocortical and corticothalamic connections of RFA and CFA differed from each other in the sense that CFA was mainly interconnected with the ventrolateral thalamic nucleus, while RFA was mainly connected with the ventromedial thalamic nucleus. Altogether, these connectional differences, compared with the pattern of organization of the motor cortical areas in primates, suggest that RFA in the rat may well be an equivalent of the premotor or supplementary motor area. In contrast to the corticocortical, corticostriatal, and thalamocortical connections, RFA and CFA showed similar efferent projections to the subthalamic nucleus, substantia nigra, red nucleus, tectum, pontine nuclei, inferior olive, and spinal cord.

Differential expression of γ-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of γ‐aminobutyric acid type B receptor (GABABR)‐ mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non‐GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR‐mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR‐mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR‐mediated disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000.

Dexterity in adult monkeys following early lesion of the motor cortical hand area: the role of cortex adjacent to the lesion

Infant monkeys were subjected to unilateral lesions of the motor cortex (mainly its hand representation). After maturation, they showed normal use of the contralateral hand for global grip movements. However, as compared with the ipsilateral hand, precision grip tasks requiring relatively independent finger movements were performed with less dexterity, particularly if adjustments of the wrist position were necessary. The purpose of this study was to investigate mechanisms which may be responsible for the rather well, although not complete, preservation of manipulative behaviour of these adult monkeys. To this end, the hand representations were mapped bilaterally with intracortical microstimulation in the mature monkeys, and the dexterity of both hands assessed quantitatively in a precision grip task. The behavioural effects of reversible inactivations of the primary (M1) and supplementary (SMA) motor cortical areas were then tested. The following were found. (i) The hand contralateral to the lesion exhibited subtle but significant dexterity deficits, as compared with the ipsilateral hand; the deficit was essentially for complex movements requiring dissociation of the thumb–index finger pinch from the other digits, involving also an arm rotation. (ii) Reversible inactivation of the M1 hand representation in the intact hemisphere dramatically impaired dexterity of the opposite hand without affecting the ipsilateral hand (contralateral to the early lesion). (iii) A relatively complete hand representation was found to occupy a new territory, medial to the old lesion. (iv) The role of this new displaced representation was crucial for the preserved dexterity of the opposite hand, as evidenced by its functional inactivation. In contrast, inactivation of both SMA cortices did not interfere with the manipulative behaviour. It is thus concluded that the preserved functional capacity of manipulations with the hand opposite the early lesion can be essentially attributed to a cortical reorganization around the old lesion. Under the present experimental conditions, contributions from either the SMA or the intact M1 appear not to be crucial.

Mapping of c-fos expression elicited by pure tones stimulation in the auditory pathways of the rat, with emphasis on the cochlear nucleus

C-fos expression was mapped in the auditory pathways of rats, stimulated acoustically with pure tones. In the cochlear nucleus, two clusters of c-fos-like immunoreactive neurons, located respectively in the caudal part of the dorsal cochlear nucleus and in the granular cell region, did not show clear systematic shift in their position as a function of the tones frequency. On the other hand, more rostrally in the dorsal cochlear nucleus, a cluster of c-fos-like positive neurons moved progressively from dorsal to ventral for decreasing tones frequency. In the posteroventral cochlear nucleus, another cluster of c-fos-like positive neurons was observed, whose position also varied with tones frequency. Surprisingly, no or very rare c-fos-like immunoreactive neurons were present in the anteroventral cochlear nucleus and in the superior olivary complex. In the inferior colliculus, however, c-fos-like immunoreactive neurons formed clear isofrequency contours, shifting from dorsolateral to ventromedial for increasing tones frequency. In the medial geniculate body c-fos-like immunostaining was restricted to the medial and dorsal divisions while the ventral division was free of labeling. The cause of this differential labeling along the auditory pathways is at present unknown but may eventually provide clues as to physiological differences in parallel auditory pathways.

Mapping of the motor pathways in rats: c-fos induction by intracortical microstimulation of the motor cortex correlated with efferent connectivity of the site of cortical stimulation

The general goal of the present study was to investigate structural components of a neural system anatomically as well as functionally. The rat motor system, which is reasonably well understood, was selected and a new procedure was developed to combine a functional marker with axonal tracing methods (in the same animal). This was achieved by mapping c-fos induction immunocytochemically as a result of intracortical microstimulation in the distal forelimb area of the motor cortex. The anterograde tracers Phaseolus vulgaris-leucoagglutinin or biocytin were deposited at the site of intracortical microstimulation, the former three weeks and the latter two to three days before stimulation. Neuronal nuclei, labeled for the expressed c-fos protein, were present and mapped in the following structures: motor cortex; basal ganglia (caudate-putamen, globus pallidus); thalamus (reticular, ventromedial and posterior nuclei); subthalamic nucleus; substantia nigra; tectum; red nucleus; pontine nuclei; inferior olive; external cuneate nucleus; cerebellar cortex; deep cerebellar nuclei. Labeling was often bilateral but generally more substantial ipsilaterally, except in the cerebellum where it was mainly contralateral. Axonal labeling, including terminal branches and boutons, was also found in most of the above structures with the exception of the globus pallidus, deep cerebellar nuclei, cerebellar cortex and external cuneate nucleus. These expected exceptions demonstrate that activity changes in these latter structures, as revealed by c-fos labeled neurons, were induced over more than one synapse. This combined procedure might, therefore, be useful in deciding whether two structures in a given system are linked directly (monosynaptically) or indirectly (polysynaptically) to each other. In contrast to the 2-deoxyglucose technique, functional mapping by means of c-fos induction provides cellular resolution, making it possible to establish fine details of axonal contacts with target neurons: boutons in close apposition to c-fos labeled neurons were clearly observed here, for instance in the cerebral cortex, caudate-putamen, thalamus, subthalamic nucleus and pontine nuclei. Surprisingly, the ventrolateral and ventrobasalis nuclei of the thalamus contained numerous and dense axon terminals labeled with Phaseolus vulgaris-leucoagglutinin or biocytin, but the contacted neurons in the ventrolateral and ventrobasalis nuclei were not marked with c-fos. However, with respect to directly connected structures, there was, in general, a good correlation between structures with axonal labeling and those with c-fos labeled neurons.

The occ1 gene is preferentially expressed in the primary visual cortex in an activity‐dependent manner: a pattern of gene expression related to the cytoarchitectonic area in adult macaque neocortex

Marker molecules to visualize specific subsets of neurons are useful for studying the functional organization of the neocortex. One approach to identify such molecular markers is to examine the differences in molecular properties among morphologically and physiologically distinct neuronal cell types. We used differential display to compare mRNA expression in the anatomically and functionally distinct areas of the adult macaque neocortex. We found that a gene, designated occ1, was preferentially transcribed in the posterior region of the neocortex, especially in area 17. Complete sequence analysis revealed that occ1 encodes a macaque homolog of a secretable protein, TSC‐36/follistatin‐related protein (FRP). In situ hybridization histochemistry confirmed the characteristic neocortical expression pattern of occ1 and showed that occ1 transcription is high in layers II, III, IVA and IVC of area 17. In addition, occ1 transcription was observed selectively in cells of the magnocellular layers in the lateral geniculate nucleus (LGN). Dual labeling immunohistochemistry showed that the occ1‐positive neurons in area 17 include both γ‐aminobutyric acid (GABA)‐positive aspiny inhibitory cells and the α‐subunit of type II calcium/calmodulin‐dependent protein kinase (CaMKII α)‐positive spiny excitatory cells. With brief periods of monocular deprivation, the occ1 mRNA level decreased markedly in deprived ocular dominance columns of area 17. From this we conclude that the expression of occ1 mRNA is present in a subset of neurons that are preferentially localized in particular laminae of area 17 and consist of various morphological and physiological neuronal types, and, furthermore, occ1 transcription is subject to visually driven activity‐dependent regulation.

Modulation of Sustained Electromyographic Activity by Single Intracortical Microstimuli: Comparison of Two Forelimb Motor Cortical Areas of the Rat

In rats, a rostral and a caudal forelimb motor area (RFA and CFA, respectively) have been distinguished on the basis of intracortical microstimulation effects (see Neafsey et al., 1986, for a review). The goal of the present study was to assess and compare their relative connectional strength with target motor units of the forelimb. This was achieved by averaging modulation responses of sustained electromyographic (EMG) activity triggered by single intracortical microstimuli (S-ICMS) of relatively low intensity (mostly below 35 microA) to minimize both direct and transsynaptic current spread. In chronically prepared and ketamine-sedated rats, this method produced prominent peaks and troughs in the averaged EMG at short latencies with S-ICMS currents as low as 5 microA. S-ICMS at 30-50 microA in CFA sometimes even elicited visible twitches and an EMG burst of the contralateral wrist or digits following each stimulation pulse. Increasing S-ICMS currents to about 1.5 mA revealed a sudden shortening of EMG response latencies, which was most likely induced by current spread to brainstem motor centers. S-ICMS at near-threshold intensity in the majority of effective sites in both CFA and RFA produced modulation responses in more than one group of forelimb muscles, frequently also including muscles of the ipsilateral forelimb. Usually the ipsilateral responses were weaker, as were the suppression effects. Comparison of CFA and RFA revealed similar effects in terms of the number of modulated muscle groups and the response latencies. In contralateral wrist/digit muscles, facilitation responses were elicited at latencies of 9.7 +/- 1.8 msec (CFA) and 9.6 +/- 1.9 msec (RFA), with the shortest latencies around 6 msec. However, modulations by S-ICMS in RFA had significantly smaller amplitudes, had slower rates of buildup, and required higher thresholds than those obtained from S-ICMS in CFA. It is concluded, on the basis of the S-ICMS method, that both the CFA and the RFA exert a prominent and relatively direct influence on forelimb motoneurons. The present findings, together with calculations of conduction time, suggest that a contingent of corticospinal axons of the rat has oligosynaptic and possibly even monosynaptic connections with forelimb motoneurons. The recruitment of a relatively large number of muscles, including those of the ipsilateral forelimb, by S-ICMS in both areas may be explained by the prominent divergence of corticospinal axons. Further investigations are required to understand the relative positions and roles of the two areas in motor control and their possible homology with primary and nonprimary motor areas of primates.

Brain-specific BNIP-2-homology protein Caytaxin relocalises glutaminase to neurite terminals and reduces glutamate levels

Human Cayman ataxia and mouse or rat dystonia are linked to mutations in the genes ATCAY (Atcay) that encode BNIP-H or Caytaxin, a brain-specific member of the BNIP-2 family. To explore its possible role(s) in neuronal function, we used protein precipitation and matrix-assisted laser desorption/ionisation mass spectrometry and identified kidney-type glutaminase (KGA) as a novel partner of BNIP-H. KGA converts glutamine to glutamate, which could serve as an important source of neurotransmitter. Co-immunoprecipitation with specific BNIP-H antibody confirmed that endogenous BNIP-H and KGA form a physiological complex in the brain, whereas binding studies showed that they interact with each other directly. Immunohistochemistry and in situ hybridisation revealed high BNIP-H expression in hippocampus and cerebellum, broadly overlapping with the expression pattern previously reported for KGA. Significantly, BNIP-H expression was activated in differentiating neurons of the embryonic carcinoma cell line P19 whereas its overexpression in rat pheochromocytoma PC12 cells relocalised KGA from the mitochondria to neurite terminals. It also reduced the steady-state levels of glutamate by inhibiting KGA enzyme activity. These results strongly suggest that through binding to KGA, BNIP-H could regulate glutamate synthesis at synapses during neurotransmission. Thus, loss of BNIP-H function could render glutamate excitotoxicity or/and deregulated glutamatergic activation, leading to ataxia, dystonia or other neurological disorders.

Cortical influences on cervical motoneurons in the rat: recordings of synaptic responses from motoneurons and compound action potential from corticospinal axons

The synaptic responses of cervical motoneurons to intracortical stimulation (ICS) of the motor cortex were studied in the rat by means of intracellular recordings. Motoneurons (n = 80) were identified either by their antidromic response to peripheral nerve electrical stimulation and/or by intracellular staining with biocytin. As a result of ICS (0.6-1.5 mA) of the contralateral motor cortex, the vast majority of motoneurons responded with EPSPs (77 out of 80), while only three motoneurons exhibited IPSPs. For increasing ICS intensities, the amplitude of the EPSPs in a given motoneuron increased, whereas their latency was not substantially affected. For the whole population of motoneurons, identified mainly by their antidromic response, the latency of the EPSPs was on average 8.45 ms (SD 1.6 ms), ranging from 4.7 to 12.6 ms. A very comparable latency distribution was obtained from the subpopulation of biocytin stained motoneurons (n = 23). In 7 of 19 tested motoneurons EPSPs could follow high frequencies (50-100 Hz) of stimulation without change of latency. The compound action potential (descending volley) travelling along corticospinal fibers reached the level of intracellular recording with a minimal latency estimated to be about 3 ms after ICS. The conduction velocity of corticospinal axons contributing to the descending volley was calculated to range from 9 to 19.7 m/s, based on morphometric measurements of conduction distance from the motor cortex and duration of the compound action potential. The time delay between the latency of descending volley and the latency of early EPSPs on the one hand, and frequency following properties of EPSPs on the other hand, suggest that some cervical motoneurons receive secure, most likely, indirect (presumably disynaptic) inputs from fast conducting corticospinal axons or direct contacts from slower conducting corticospinal fibers. The biocytin labeled cervical motoneurons exhibited extraordinary long dendritic trees, extending both laterally in the white matter near the edge of the spinal cord and medially in the gray matter as far as the midline of the spinal cord. The motoneurons were also characterized by the presence of one or several recurrent axon collaterals, ramifying profusely in the neuropil, with numerous boutons en passant and terminaux contacting most likely neighboring cervical neurons.