Pain (nociceptive) input caudal to a spinal contusion injury increases tissue loss and impairs long-term recovery. It was hypothesized that noxious stimulation has this effect because it engages unmyelinated pain (C) fibers that produce a state of over-excitation in central pathways. The present article explored this issue by assessing the effect of capsaicin, which activates C-fibers that express the transient receptor potential vanilloid receptor-1 (TRPV1). Rats received a lower thoracic (T11) contusion injury and capsaicin was applied to one hind paw the next day. For comparison, other animals received noxious electrical stimulation at an intensity that engages C fibers. Both forms of stimulation elicited similar levels of c-fos mRNA expression, a cellular marker of nociceptive activation, and impaired long-term behavioral recovery. Cellular assays were then performed to compare the acute effect of shock and capsaicin treatment. Both forms of noxious stimulation increased expression of tumor necrosis factor (TNF) and caspase-3, which promotes apoptotic cell death. Shock, but not capsaicin, enhanced expression of signals related to pyroptotic cell death [caspase-1, inteleukin-1 beta (IL-1ß)]. Pyroptosis has been linked to the activation of the P2X7 receptor and the outward flow of adenosine triphosphate (ATP) through the pannexin-1 channel. Blocking the P2X7 receptor with Brilliant Blue G (BBG) reduced the expression of signals related to pyroptotic cell death in contused rats that had received shock. Blocking the pannexin-1 channel with probenecid paradoxically had the opposite effect. BBG enhanced long-term recovery and lowered reactivity to mechanical stimulation applied to the girdle region (an index of chronic pain), but did not block the adverse effect of nociceptive stimulation. The results suggest that C-fiber input after injury impairs long-term recovery and that this effect may arise because it induces apoptotic cell death.
Lamina I is a sensory relay region containing projection cells and local interneurons involved in thermal and nociceptive signaling. These neurons differ in morphology, sensory response modality, and firing characteristics. We examined intrinsic properties of mouse lamina I GABAergic neurons expressing enhanced green fluorescent protein (EGFP). GABAergic neuron identity was confirmed by a high correspondence between GABA immunolabeling and EGFP fluorescence. Morphologies of these EGFP+/GABA+ cells were multipolar (65%), fusiform (31%), and pyramidal (4%). In whole cell recordings, cells fired a single spike (44%), tonically (35%), or an initial burst (21%) in response to current steps, representing a subset of reported lamina I firing properties. Membrane properties of tonic and initial burst cells were indistinguishable and these neurons may represent one functional population because, in individual neurons, their firing patterns could interconvert. Single spike cells were less excitable with lower membrane resistivity and higher rheobase. Most fusiform cells (64%) fired tonically while most multipolar cells (56%) fired single spikes. In summary, lamina I inhibitory interneurons are functionally divisible into at least two major groups both of which presumably function to limit excitatory transmission.
When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABAA transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABAA transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABAA receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABAA receptor. Therefore, GABA appears to be serving as a proxy for activity levels.
Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2C receptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague-Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor-mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2C and 5-HT1A receptor agonists, respectively, regardless of substrain. LLFR was predominantly Aβ afferent fiber mediated, consistent with prominent 5-HT2C receptor expression in the Aβ fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor-mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAA and/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors.
Available evidence supports the idea that muscle fibres provide retrograde signals that enable the expression of adult motoneuron electrical properties but the mechanisms remain unknown. We showed recently that when acetylcholine receptors are blocked at motor endplates, the electrical properties of rat motoneurons change in a way that resembles changes observed after axotomy. This observation suggests that receptor blockade and axotomy interrupt the same signalling mechanisms but leaves open the possibility that the loss of muscle fibre activity underlies the observed effects. To address this issue, we examined the electrical properties of axotomized motoneurons following reinnervation. Ordinarily, these properties return to normal following reinnervation and re-activation of muscle, but in this study muscle fibre activity and evoked acetylcholine release were prevented during reinnervation by blocking axonal conduction. Under these conditions, the properties of motoneurons that successfully reinnervated muscle fibres recovered to normal despite the absence of muscle fibre activity and evoked release. We conclude that the expression of motoneuron electrical properties is not regulated by muscle fibre activity but rather by a retrograde signalling system coupled to activation of endplate acetylcholine receptors. Our results indicate that spontaneous release of acetylcholine from regenerated motor terminals is sufficient to operate the system.
The electrogenic Na+–HCO3− cotransporter (NBCe1) plays a central role in intracellular pH (pHi) regulation as well as HCO3− secretion by pancreatic ducts and HCO3− reabsorption by renal proximal tubules. To understand the structural requirements for the electrogenicity of NBCe1, we constructed chimeras of NBCe1-A and the electroneutral NBCn1-B, and used two-electrode voltage clamp to measure electrogenic transporter current in Xenopus oocytes exposed to 5% CO2–26 mm HCO3−(pH 7.40). The chimera consisting of NBCe1-A (i.e. NBCe1-A ‘background’) with the cytoplasmic N-terminal domain (Nt) of NBCn1-B had a reversal potential of −156.3 mV (compared with a membrane potential Vm of −43.1 mV in a HCO3−-free solution) and a slope conductance of 3.0 μS (compared with 12.5 μS for NBCe1-A). Also electrogenic were chimeras with an NBCe1-A background but with NBCn1-B contributing the extracellular loop (L) between transmembrane segment (TM) 5 and 6 (−140.9 mV/11.1 μS), the cytoplasmic C-terminal domain (Ct; −123.8 mV/9.7 μS) or Nt + L + Ct (−120.9 mV/3.7 μS). Reciprocal chimeras (with an NBCn1 background but with NBCe1 contributing Nt, L, Ct or Nt + L + Ct) produced no measurable electrogenic transporter currents in the presence of CO2–HCO3−. pHi recovered from an acid load, but without the negative shift of Vm that is characteristic of electrogenic Na+–HCO3− cotransporters. Thus, these chimeras were electroneutral, as were two others consisting of NBCe1(Nt–L)/NBCn1(TM6–Ct) and NBCn1(Nt–L)/NBCe1(TM6–Ct). We propose that the electrogenicity of NBCe1 requires interactions between TM1–5 and TM6–13.
Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan-treated rats (0.34 ± 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 ± 0.37 nM) that were decreased by candesartan (0.97 ± 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 ± 13%) and base (120 ± 25%). UT-A3 abundance was increased in IM tip (123 ± 11%) and base (146 ± 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 ± 14%) and base (210 ± 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 ± 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM.
Recent studies have shown that accessory proteins that interact with the apical Na+/H+ exchanger NHE3 are a vital part of the dynamic nature of the Na+/H+ exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism.
During early development, γ-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (EGABA) for GABAA miniature postsynaptic currents. When whole cell patch solutions contained 17–52 mM chloride, we found that synaptic EGABA was around −30 mV. Because of the low HCO3− permeability of the GABAA receptor, this value of EGABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na+-K+-2Cl− cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized EGABA and reduced nerve potentials evoked by local application of a GABAA agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO3− and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.
The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH2O. In contrast, urine osmolality was low (800 mosmol/kgH2O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.