SYNAPTIC PLASTICITY IN THE INJURED BRAIN DEPENDS ON THE TEMPORAL PATTERN OF STIMULATION.

Neurostimulation protocols are increasingly used as therapeutic interventions, including for brain injury. In addition to the direct activation of neurons, these stimulation protocols are also likely to have downstream effects on those neurons' synaptic outputs. It is well known that alterations in the strength of synaptic connections (long-term potentiation, LTP; long-term depression, LTD) are sensitive to the frequency of stimulation used for induction, however little is known about the contribution of the temporal pattern of stimulation to the downstream synaptic plasticity that may be induced by neurostimulation in the injured brain. We explored interactions of the temporal pattern and frequency of neurostimulation in the normal cerebral cortex and after mild traumatic brain injury (mTBI), to inform therapies to strengthen or weaken neural circuits in injured brains, as well as to better understand the role of these factors in normal brain plasticity. Whole-cell (WC) patch-clamp recordings of evoked postsynaptic potentials (PSPs) in individual neurons, as well as field potential (FP) recordings, were made from layer 2/3 of visual cortex in response to stimulation of layer 4, in acute slices from control (naïve), sham operated, and mTBI rats. We compared synaptic plasticity induced by different stimulation protocols, each consisting of a specific frequency (1 Hz, 10 Hz, or 100 Hz), continuity (continuous or discontinuous), and temporal pattern (perfectly regular, slightly irregular, or highly irregular). At the individual neuron level, dramatic differences in plasticity outcome occurred when the highly irregular stimulation protocol was used at 1 Hz or 10 Hz, producing an overall LTD in controls and shams, but a robust overall LTP after mTBI. Consistent with the individual neuron results, the plasticity outcomes for simultaneous FP recordings were similar, indicative of our results generalizing to a larger scale synaptic network than can be sampled by individual WC recordings alone. In addition to the differences in plasticity outcome between control (naïve or sham) and injured brains, the dynamics of the changes in synaptic responses that developed during stimulation were predictive of the final plasticity outcome. Our results demonstrate that the temporal pattern of stimulation plays a role in the polarity and magnitude of synaptic plasticity induced in the cerebral cortex while highlighting differences between normal and injured brain responses. Moreover, these results may be useful for optimization of neurostimulation therapies to treat mTBI and other brain disorders, in addition to providing new insights into downstream plasticity signaling mechanisms in the normal brain.

Sirt1 ablation promotes stress-induced loss of epigenetic and genomic hematopoietic stem and progenitor cell maintenance.

The (histone) deacetylase Sirt1 is a mediator of genomic and epigenetic maintenance, both of which are critical aspects of stem cell homeostasis and tightly linked to their functional decline in aging and disease. We show that Sirt1 ablation in adult hematopoietic stem and progenitor cells (HSPCs) promotes aberrant HSPC expansion specifically under conditions of hematopoietic stress, which is associated with genomic instability as well as the accumulation of DNA damage and eventually results in a loss of long-term progenitors. We further demonstrate that progenitor cell expansion is mechanistically linked to the selective up-regulation of the HSPC maintenance factor and polycomb target gene Hoxa9. We show that Sirt1 binds to the Hoxa9 gene, counteracts acetylation of its histone target H4 lysine 16, and in turn promotes polycomb-specific repressive histone modification. Together, these findings demonstrate a dual role for Sirt1 in HSPC homeostasis, both via epigenetic regulation of a key developmental gene and by promoting genome stability in adult stem cells.

A residue in loop 9 of the beta2-subunit stabilizes the closed state of the GABAA receptor.

In gamma-aminobutyric acid type A (GABA(A)) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the beta2-subunit may be involved in GABA(A) receptor activation. Specifically, residues Gly(170)-Gln(185) of the beta2-subunit were mutated to alanine, co-expressed with wild-type alpha1- and gamma2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC(50) whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC(50). None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC(50). Taken together, these results indicate that beta2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that beta2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA(A) receptor.

Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit.

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of alpha and beta subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the gamma subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered gamma2(L287A) mutation on GABA activation and propofol modulation. In the macroscopic analysis, we found that the gamma2(L287A) mutation decreased GABA potency but increased the ability of propofol to enhance both GABA potency and efficacy compared with wild-type receptors. Indeed, although propofol had significant effects on GABA potency in wild-type receptors, we found that propofol produced no corresponding increase in GABA efficacy. At the single-channel level, mutant receptors showed a loss in the longest of three open-time components compared with wild-type receptors under GABA activation. Furthermore, propofol reduced the duration of one closed-time component, increased the duration of two open-time components, and generated a third open component with a longer lifetime in mutant compared with wild-type receptors. Taken together, we conclude that although the gamma subunit is not required for the binding of GABA or propofol, the M2-M3 linker of this subunit plays a critical role in channel gating by GABA and allosteric modulation by propofol. Our results also suggest that in wild-type receptors, propofol exerts its enhancing effects by mechanisms extrinsic to channel gating.

A comparison of resonance tuning with positive versus negative sensory feedback.

We used a computational model of rhythmic movement to analyze how the connectivity of sensory feedback affects the tuning of a closed-loop neuromechanical system to the mechanical resonant frequency (omega(r)). Our model includes a Matsuoka half-center oscillator for a central pattern generator (CPG) and a linear, one-degree-of-freedom system for a mechanical component. Using both an open-loop frequency response analysis and closed-loop simulations, we compared resonance tuning with four different feedback configurations as the mechanical resonant frequency, feedback gain, and mechanical damping varied. The feedback configurations consisted of two negative and two positive feedback connectivity schemes. We found that with negative feedback, resonance tuning predominantly occurred when omega(r) was higher than the CPG's endogenous frequency (omega(CPG)). In contrast, with the two positive feedback configurations, resonance tuning only occurred if omega(r) was lower than omega(CPG). Moreover, the differences in resonance tuning between the two positive (negative) feedback configurations increased with increasing feedback gain and with decreasing mechanical damping. Our results indicate that resonance tuning can be achieved with positive feedback. Furthermore, we have shown that the feedback configuration affects the parameter space over which the endogenous frequency of the CPG or resonant frequency the mechanical dynamics dominates the frequency of a rhythmic movement.

Resonance tuning of a neuromechanical system with two negative sensory feedback configurations.

Resonance tuning in a model of rhythmic movement is compared when the central pattern generator (CPG) consists of two endogenously bursting or two tonically spiking neurons that are connected with reciprocally inhibitory synapses. The CPG receives inhibitory and/or excitatory position feedback from a linear, one-degree-of-freedom mechanical subsystem. As with previously published results [5, 15], resonance tuning is limited to frequencies that are greater than the intrinsic CPG frequency with endogenously bursting neurons. In contrast, with tonically spiking neurons, the resonance tuning range is expanded to frequencies that are below the intrinsic CPG frequency.