Although mean diffusivity and fractional anisotropy abnormalities in these patients with TBI were too subtle to be detected with the whole-brain histogram analysis, they are present in brain areas that are frequent sites of DAI. Because diffusion tensor imaging changes are present at both early and late time points following injury, they may represent an early indicator and a prognostic measure of subsequent brain damage.

Synucleins are a vertebrate-specific family of abundant neuronal proteins. They comprise three closely related members, α-, β-, and γ-synuclein. α-Synuclein has been the focus of intense attention since mutations in it were identified as a cause for familial Parkinson's disease. Despite their disease relevance, the normal physiological function of synucleins has remained elusive. To address this, we generated and characterized αβγ-synuclein knockout mice, which lack all members of this protein family. Deletion of synucleins causes alterations in synaptic structure and transmission, age-dependent neuronal dysfunction, as well as diminished survival. Abrogation of synuclein expression decreased excitatory synapse size by ∼30% both in vivo and in vitro, revealing that synucleins are important determinants of presynaptic terminal size. Young synuclein null mice show improved basic transmission, whereas older mice show a pronounced decrement. The late onset phenotypes in synuclein null mice were not due to a loss of synapses or neurons but rather reflect specific changes in synaptic protein composition and axonal structure. Our results demonstrate that synucleins contribute importantly to the long-term operation of the nervous system and that alterations in their physiological function could contribute to the development of Parkinson's disease.neurodegeneration | loss-of-function | Lewy bodies | ultrastructure | retina S ynucleins are a family of vertebrate-specific proteins with three closely related members, α-, β-, and γ-synuclein (1, 2). They are abundant neuronal proteins and are reported to account for 0.1% of total brain protein (3, 4). Synucleins have overlapping expression patterns and are enriched in presynaptic termini (5, 6). α-Synuclein has been the focus of intense attention since the identification of dominant mutations and gene multiplications that link it to familial Parkinson's disease (PD) (7). Recently, strong ties between the α-synuclein gene and sporadic PD have emerged in genomewide association studies, making α-synuclein the most broadly relevant PD gene (8, 9). Additionally, α-synuclein is the major component of Lewy bodies, the pathological hallmark of PD (10). The presynaptic localization and function of synucleins may also have a bearing on PD, as synapses are lost early in disease progression (11).Analysis of α-, β-, and γ-synuclein sequences reveals a shared, highly conserved N-terminal domain (∼80% identical) with a less conserved acidic C terminus (2). The N-terminal domain has seven imperfect repeats of 11 residues with the consensus sequence XKTKEGVXXXX that binds acidic phospholipid surfaces. Upon lipid binding, synucleins undergo a dramatic change to an α-helical conformation (12-14). α-Synuclein adopts either a conformation consisting of two anti-parallel, amphipathic α-helices with an unfolded C terminus (13-15) or a single extended α-helical structure (16, 17). Human β-and γ-synuclein also adopt the two-helix conformation upon folding (18,19). Together, the high sequence homolog...

Genetic and pathological studies link ␣-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. ␣-Synuclein, an acidic lipid binding protein, shares high sequence identity with ␤-and ␥-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse ␣-, ␤-, or ␥-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knock-out synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize ␣-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.

Previous studies have implicated extracellular carbonic anhydrases (CAs) in buffering the alkaline pH shifts that accompany neuronal activity in the rat and mouse hippocampus. CAs IV and XIV both have been proposed to mediate this extracellular buffering. To examine the relative importance of these two isozymes in this and other physiological functions attributed to extracellular CAs, we produced CA IV and CA XIV knockout (KO) mice by targeted mutagenesis and the doubly deficient CA IV͞XIV KO mice by intercrossing the individual null mice. Although CA IV and CA XIV null mice both are viable, the CA IV nulls are produced in smaller numbers than predicted, indicating either fetal or postnatal losses, which preferentially affect females. CA IV͞XIV double KO mice are also produced in fewer numbers than predicted and are smaller than WT mice, and many females die prematurely before and after weaning. Electrophysiological studies on hippocampal slices on these KO mice showed that either CA can mediate buffering after synaptic transmission in hippocampal slices in the absence of the other, but that eliminating both is nearly as effective as the CA inhibitor, benzolamide, in blocking the buffering seen in the WT mice. Thus, both CA IV and CA XIV contribute to extracellular buffering in the central nervous system, although CA IV appears to be more important in the hippocampus. These individual and double KO mice should be valuable tools in clarifying the relative contributions of each CA to other physiological functions where extracellular CAs have been implicated. interstitial pH ͉ mouse hippocampus ͉ targeted mutagenesis

Makani S, Chesler M. Rapid rise of extracellular pH evoked by neural activity is generated by the plasma membrane calcium ATPase. J Neurophysiol 103: 667-676, 2010. First published November 25, 2009 doi:10.1152/jn.00948.2009. In hippocampus, synchronous activation of CA1 pyramidal neurons causes a rapid, extracellular, population alkaline transient (PAT). It has been suggested that the plasma membrane Ca 2ϩ -ATPase (PMCA) is the source of this alkalinization, because it exchanges cytosolic Ca 2ϩ for external H ϩ . Evidence supporting this hypothesis, however, has thus far been inconclusive. We addressed this long-standing problem by measuring surface alkaline transients (SATs) from voltage-clamped CA1 pyramidal neurons in juvenile mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes placed against the somata. In saline containing benzolamide (a poorly permeant carbonic anhydrase blocker), a 2-s step from Ϫ60 to 0 mV caused a mean SAT of 0.02 unit pH. Addition of 5 mM HEPES to the artificial cerebrospinal fluid diminished the SAT by 91%. Nifedipine reduced the SAT by 53%. Removal of Ca 2ϩ from the saline abolished the SAT, and addition of BAPTA to the patch pipette reduced it by 79%. The inclusion of carboxyeosin (a PMCA inhibitor) in the pipette abolished the SAT, whether it was induced by a depolarizing step, or by simulated, repetitive, antidromic firing. The peak amplitude of the "antidromic" SAT of a single cell averaged 11% of the PAT elicited by comparable real antidromic activation of the CA1 neuronal population. Caloxin 2A1, an extracellular PMCA peptide inhibitor, blocked both the SAT and PAT by 42%. These results provide the first direct evidence that the PMCA can explain the extracellular alkaline shift elicited by synchronous firing.

In hippocampus, activation of the Schaffer collaterals generates an extracellular alkaline transient both in vitro and in vivo. This pH change may provide relief of the H ϩ block of NMDA receptors (NMDARs) and thereby increase excitability. To test this hypothesis, we augmented extracellular buffering in mouse hippocampal slices by adding 2 M bovine type II carbonic anhydrase to the superfusate. With addition of enzyme, the alkaline transient elicited by a 10 pulse, 100 Hz stimulus train was reduced by 33%. At a holding potential (V H ) of Ϫ30 mV, the enzyme decreased the half-time of decay and charge transfer of EPSCs by 32 and 39%, respectively, but had no effect at a V H of Ϫ80 mV. In current clamp, a 10 pulse, 100 Hz stimulus train gave rise to an NMDAR-dependent afterdepolarization (ADP).

In the hippocampus, extracellular carbonic anhydrase (Car) speeds the buffering of an activity-generated rise in extracellular pH that impacts H+-sensitive N-methyl-D-aspartate receptors (NMDARs). We studied the role of Car14 in this brain structure, where it is expressed solely on neurons. Current-clamp responses were recorded from CA1 pyramidal neurons in wild type (WT) vs. Car14 knock out (KO) mice 2 s. before (control) and after (test) a 10 pulse, 100 Hz afferent train. In both WT and KO, the half width (HW) of the test response, and its number of spikes, were augmented relative to the control. An increase in presynaptic release was not involved, as AMPAR-mediated EPSCs were depressed after a train. The increases in HW and spike number were both greater in the Car14 KO. In 0 Mg2+ saline with picrotoxin (using a 20 Hz train), the HW measures were still greater in the KO. The Car inhibitor benzolamide (BZ) enhanced the test response HW in the WT, but had no effect on the already-prolonged HW in the KO. With intracellular MK-801, the curtailed WT and KO responses were indistinguishable, and BZ caused no change. By contrast, the extracellular alkaline changes evoked by the train were not different between WT and KO, and BZ amplified these alkalinizations similarly. These data suggest that Car14 regulates pH transients in the perisynaptic microenvironment, and govern their impact on NMDARs, but plays little role in buffering pH shifts in the broader, macroscopic, extracellular space.

In many brain regions, synchronous neural activity causes a rapid rise in extracellular pH. In the CA1 region of hippocampus, this population alkaline transient (PAT) enhances responses from postsynaptic, pH-sensitive N-methyl-d-aspartate (NMDA) receptors. Recently, we showed that the plasma membrane Ca(2+)-ATPase (PMCA), a ubiquitous transporter that exchanges internal Ca(2+) for external H(+), is largely responsible for the PAT. It has also been shown that a PAT can be generated after replacing extracellular Ca(2+) with Ba(2+). The cause of this PAT is unknown, however, because the ability of the mammalian PMCA to transport Ba(2+) is unclear. If the PMCA did not carry Ba(2+), a different alkalinizing source would have to be postulated. Here, we address this issue in mouse hippocampal slices, using concentric (high-speed, low-noise) pH microelectrodes. In Ba(2+)-containing, Ca(2+)-free artificial cerebrospinal fluid, a single antidromic shock to the alveus elicited a large (0.1-0.2 unit pH), "all-or-none" PAT in the CA1 cell body region. In whole cell current clamp of single CA1 pyramidal neurons, the same stimulus evoked a prolonged plateau potential that was similarly all-or-none. Using this plateau as the voltage command in other cells, we recorded Ba(2+)-dependent surface alkaline transients (SATs). The SATs were suppressed by adding 5 mM extracellular HEPES and abolished when carboxyeosin (a PMCA inhibitor) was in the patch pipette solution. These results suggest that the PAT evoked in the presence of Ba(2+) is caused by the PMCA and that this transporter is responsible for the PAT whether Ca(2+) or Ba(2+) is the charge carrying divalent cation.