Whether neocortical γ-aminobutyric acid (GABA) cells are composed of a limited number of distinct classes of neuron, or whether they are continuously differentiated with much higher diversity, remains a contentious issue for the field. Most GABA cells of rat frontal cortex have at least 1 of 6 chemical markers (parvalbumin, calretinin, alpha-actinin-2, somatostatin, vasoactive intestinal polypeptide, and cholecystokinin), with each chemical class comprising several distinct neuronal subtypes having specific physiological and morphological characteristics. To better clarify GABAergic neuron diversity, we assessed the colocalization of these 6 chemical markers with corticotropin-releasing factor (CRF), neuropeptide Y (NPY), the substance P receptor (SPR), and nitric oxide synthase (NOS); these 4 additional chemical markers suggested to be expressed diversely or specifically among cortical GABA cells. We further correlated morphological and physiological characteristics of identified some chemical subclasses of inhibitory neurons. Our results reveal expression specificity of CRF, NPY, SPR, and NOS in morphologically and physiologically distinct interneuron classes. These observations support the existence of a limited number of functionally distinct subtypes of GABA cells in the neocortex.

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.

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Lipopolysaccharide (LPS) administration in the rat produces a sepsis model of cholestasis which is directly associated with changes in the expression of several liver transporters.2) Among the various constitutively expressed influx (Ntcp, Oatp1, Oatp2, Oatp4, Oct1, Oat2, Oat3) and efflux transporters (Mrp2, Mrp3, Bsep, Mdr1a, Mdr1b) in rat liver, LPS down-regulates most of the influx and efflux transporters, but up-regulates some efflux transporters, including Mrp3 and Mdr1b. These changes are thought to represent a defense mechanism which protects the liver from the accumulation of endogenous compounds such as bile acid and bilirubin, as well as exogenous toxic compounds.With regard to mechanism, LPS induces the production of proinflammatory cytokines (cytokines), such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6 in Kupffer cells (KCs), and nitric oxide (NO) via the induction of NO synthase (iNOS) in KCs and hepatocytes. These cytokines are thought to be major mediators of the down-regulation of mRNA of transporters such as Ntcp, Oatp1, Oatp2, Mrp2, Mrp3, Bsep and Mdr1a, and of the up-regulation of Mdr1b. [3][4][5][6][7][8][9] Extensive studies of the molecular mechanisms of transporter regulation show that the LPS-induced downregulation of transporters is strongly affected by the preceding decrease in the quantity or function of nuclear transcription factors such as hepatocytes nuclear factors (HNF1a, HNF4a) and nuclear receptor heterodimers with retinoid X receptor (RXR)a (retinoic acid receptor (RAR))a, pregnane X receptor (PXR), farnesoid X receptor (FXR), constitutive ardrostane receptor (CAR)). 10)In contrast, relatively few studies have investigated the role of NO derived from iNOS in the regulation of hepatic transporters. Among studies to date, a regulatory role of NO on the mRNA of Oat2 was identified in in vivo experiments with LPS (1 mg/kg) and aminoguanidine (AG, 20 mg/kg, an iNOS inhibitor).11) On the contrary, AG (100 mg/kg) had no effect on LPS (4 mg/kg)-induced changes in rat liver transporter mRNA levels.2) Nevertheless, NO's effect in decreasing transcriptional activities of RXRa 12) and HNF4a 13) indicate its possible role as a mediator of transporter genes.Here, we investigated the role of iNOS-derived NO in the regulation of mRNA expression of rat liver transporters (Table 1; Ntcp, Oatp1, Oatp2, Oatp4, Oat2, Oat3, Oct1, Mrp2, Mrp3, Bsep, Mdr1a, Mdr1b) To determine the role of nitric oxide (NO) in rat liver transporter regulation, we investigated whether NO mediates lipopolysaccharide (LPS)-induced changes in transporters and their transcription factor expression using aminoguanidine (AG), an inhibitor of induced nitric oxide synthase (iNOS). We confirmed that LPS decreased mRNA levels for Ntcp, Oatp1, Oatp2, Oatp4, Oct1, Mrp2, Mdr1a and increased those for Mdr1b at 16 h after administration. AG attenuated these decreases for Ntcp, Oatp1 and Oatp4 (retinoid X receptor (RXR)a aand hepatocyte nuclear factor (HNF)4a a-dependent genes) and increase for Mdr1b (nuclear fact...