Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Dentate Granule Cells Are Hyperexcitable in the TgF344-AD Rat Model of Alzheimer's Disease.

The dentate gyrus is both a critical gatekeeper for hippocampal signal processing and one of the first brain regions to become dysfunctional in Alzheimer's disease (AD). Accordingly, the appropriate balance of excitation and inhibition through the dentate is a compelling target for mechanistic investigation and therapeutic intervention in early AD. Previously, we reported an increased long-term potentiation (LTP) magnitude at medial perforant path-dentate granule cell (MPP-DGC) synapses in slices from both male and acutely ovariectomized female TgF344-AD rats compared with wild type (Wt) as early as 6 months of age that is accompanied by an increase in steady-state postsynaptic depolarization during the high-frequency stimulation used to induce plasticity. Subsequently, we found that heightened function of ß-adrenergic receptors (ß-ARs) drives the increase in the LTP magnitude, but the increase in steady-state depolarization was only partially due to ß-AR activation. As we previously reported no detectable difference in spine density or presynaptic release probability, we entertained the possibility that DGCs themselves might have modified passive or active membrane properties, which may contribute to the significant increase in charge transfer during high-frequency stimulation. Using brain slice electrophysiology from 6-month-old female rats acutely ovariectomized to eliminate variability due to fluctuating plasma estradiol, we found significant changes in passive membrane properties and active membrane properties leading to increased DGC excitability in TgF344-AD rats. Specifically, TgF344-AD DGCs have an increased input resistance and decreased rheobase, decreased sag, and increased action potential (AP) spike accommodation. Importantly, we found that for the same amount of depolarizing current injection, DGCs from TgF344-AD compared with Wt rats have a larger magnitude voltage response, which was accompanied by a decreased delay to fire the first action potential, indicating TgF344-AD DGCs membranes are more excitable. Taken together, DGCs in TgF344-AD rats are more excitable, which likely contributes to the heightened depolarization during high-frequency synaptic activation.

Parvalbumin subtypes of cerebellar Purkinje cells contribute to differential intrinsic firing properties.

Purkinje cells (PCs) are central to cerebellar information coding and appreciation for the diversity of their firing patterns and molecular profiles is growing. Heterogeneous subpopulations of PCs have been identified that display differences in intrinsic firing properties without clear mechanistic insight into what underlies the divergence in firing parameters. Although long used as a general PC marker, we report that the calcium binding protein parvalbumin labels a subpopulation of PCs, based on high and low expression, with a conserved distribution pattern across the animals examined. We trained a convolutional neural network to recognize the parvalbumin subtypes and create maps of whole cerebellar distribution and find that PCs within these areas have differences in spontaneous firing that can be modified by altering calcium buffer content. These subtypes also show differential responses to potassium and calcium channel blockade, suggesting a mechanistic role for variability in PC intrinsic firing through differences in ion channel composition. It is proposed that ion channels drive the diversity in PC intrinsic firing phenotype and parvalbumin calcium buffering provides capacity for the highest firing rates observed. These findings open new avenues for detailed classification of PC subtypes.

Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function.

Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.

Deficits in synaptic function occur at medial perforant path-dentate granule cell synapses prior to Schaffer collateral-CA1 pyramidal cell synapses in the novel TgF344-Alzheimer's Disease Rat Model.

Alzheimer's disease (AD) pathology begins decades prior to onset of clinical symptoms, and the entorhinal cortex and hippocampus are among the first and most extensively impacted brain regions. The TgF344-AD rat model, which more fully recapitulates human AD pathology in an age-dependent manner, is a next generation preclinical rodent model for understanding pathophysiological processes underlying the earliest stages of AD (Cohen et al., 2013). Whether synaptic alterations occur in hippocampus prior to reported learning and memory deficit is not known. Furthermore, it is not known if specific hippocampal synapses are differentially affected by progressing AD pathology, or if synaptic deficits begin to appear at the same age in males and females in this preclinical model. Here, we investigated the time-course of synaptic changes in basal transmission, paired-pulse ratio, as an indirect measure of presynaptic release probability, long-term potentiation (LTP), and dendritic spine density at two hippocampal synapses in male and ovariectomized female TgF344-AD rats and wildtype littermates, prior to reported behavioral deficits. Decreased basal synaptic transmission begins at medial perforant path-dentate granule cell (MPP-DGC) synapses prior to Schaffer-collateral-CA1 (CA3-CA1) synapses, in the absence of a change in paired-pulse ratio (PPR) or dendritic spine density. N-methyl-d-aspartate receptor (NMDAR)-dependent LTP magnitude is unaffected at CA3-CA1 synapses at 6, 9, and 12months of age, but is significantly increased at MPP-DGC synapses in TgF344-AD rats at 6months only. Sex differences were only observed at CA3-CA1 synapses where the decrease in basal transmission occurs at a younger age in males versus females. These are the first studies to define presymptomatic alterations in hippocampal synaptic transmission in the TgF344-AD rat model. The time course of altered synaptic transmission mimics the spread of pathology through hippocampus in human AD and provides support for this model as a valuable preclinical tool in elucidating pathological mechanisms of early synapse dysfunction in AD.

17ß estradiol recruits GluN2B-containing NMDARs and ERK during induction of long-term potentiation at temporoammonic-CA1 synapses.

When circulating 17ß estradiol (E2) is elevated to proestrous levels, hippocampus-dependent learning and memory is enhanced in female rodents, nonhuman primates, and women due to heightened synaptic function at hippocampal synapses. We previously reported that proestrous-like levels of E2 administered to young adult ovariectomized (OVX) female rats increases the magnitude of LTP at CA3 Schaffer collateral (SC)-CA1 synapses only when dendritic spine density, the NMDAR/AMPAR ratio, and current mediated by GluN2B-containing NMDA receptors (NMDARs) are simultaneously increased. We also reported that this increase in GluN2B-mediated NMDAR current in area CA1 is causally related to the E2-induced increase in novel object recognition, tying together heightened synaptic function with improved learning and memory. In addition to SC inputs, innervation from the entorhinal cortex in the temporoammonic (TA) pathway onto CA1 distal dendrites in stratum lacunosum-moleculare is critical for spatial memory formation and retrieval. It is not known whether E2 modulates TA-CA1 synapses similarly to SC-CA1 synapses. Here, we report that 24 hours post-E2 injection, dendritic spine density on CA1 pyramidal cell distal dendrites and current mediated by GluN2B-containing NMDARs at TA-CA1 synapses is increased, similarly to our previous findings at SC-CA1 synapses. However, in contrast to SC-CA1 synapses, AMPAR transmission at TA-CA1 synapses is significantly increased, and there is no effect on the LTP magnitude. Pharmacological blockade of GluN2B-containing NMDARs or ERK activation, which occurs downstream of synaptic but not extrasynaptic GluN2B-containing NMDARs, attenuates the LTP magnitude only in slices from E2-treated rats. These data show that E2 recruits a causal role for GluN2B-containing NMDARs and ERK signaling in the induction of LTP, cellular mechanisms not required for LTP induction at TA-CA1 synapses in vehicle-treated OVX female rats.