Effects of sustained proNGF blockade on attentional capacities in aged rats with compromised cholinergic system

Abstract

Disruption in nerve growth factor (NGF) signaling via tropomyosin-related kinase A (trkA) receptors compromises the integrity of the basal forebrain (BF) cholinergic system, yielding cognitive, specifically attentional, impairments in Alzheimer's disease (AD). Although normal aging is considered a risk factor for AD, the mechanisms underlying the selective vulnerability of the aging cholinergic system to trkA disruption is not clear. The levels of proNGF, a proneurotrophin that possesses higher affinity for p75 receptors, increase in aging. The present study was designed to test the hypothesis that cholinergic and attentional dysfunction in aged rats with reduced BF trkA receptors occurs due to the overactivation of endogenous proNGF signaling. We employed a viral vector that produced trkA shRNA to suppress trkA receptors in the corticopetal cholinergic neurons of aged rats. BF trkA suppression impaired animals' performance on signal trials in both the sustained attention task (SAT) and the cognitively taxing distractor version of SAT (dSAT) and these deficits were normalized by chronic intracerebroventricular administration of proNGF antibody. Moreover, depolarization-evoked acetylcholine (ACh) release and the density of cortical cholinergic fibers were partially restored in these animals. However, SAT/dSAT scores reflecting overall performance did not improve following proNGF blockade in trkA knockdown rats due to impaired performance in non-signal trials. Sustained proNGF blockade alone did not alter baseline attentional performance but produced moderate impairments during challenging conditions. Collectively, our findings indicate that barring proNGF-p75 signaling may exert some beneficial effects on attentional capacities specifically when BF trkA signaling is abrogated. However, endogenous proNGF may also possess neurotrophic effects and blockade of this proneurotrophin may not completely ameliorate attentional impairments in AD and potentially hinder performance during periods of high cognitive load in normal aging.

Keywords: Alzheimer’s disease; acetylcholine; aging; attention; proNGF.

Figure 1

Schematic representation of the experimental design. After attaining criterion on SAT (see Section 2 Experimental Procedures for details), rats underwent stereotaxic surgeries for infusions of AAV vectors expressing either trkA shRNA (AAV-trkA) or shRNA for the functional control gene luciferase (AAV-luc; control vector) into the basal forebrain (BF). Simultaneously, mini-osmotic pumps connected to brain infusion cannula were implanted subcutaneously for chronic intracranial administration of either proNGF antibody (Ab) or vehicle (0.01M PBS) into the right ventricle. Animals were randomly assigned into four groups (AAV-luc + vehicle, AAV-trkA + vehicle, AAV-luc + proNGF Ab, and AAV-trkA + proNGF Ab). After recovery from surgery, rats were placed back on task up to 4 weeks for SAT/dSAT testing. At the completion of behavioral testing, animals were anesthetized to conduct electrochemical recordings of cholinergic transmission, following which they were perfused for immunohistochemical analysis.

Figure 1

Cholinergic targeting of AAV vectors, trkA knockdown and proNGF blockade. (A) Schematic representation of the AAV vector infusion site. AAV vectors expressing shRNA either for trkA (AAV-trkA) or functional control protein luciferase (AAV-luc) were infused into the nucleus basalis of Meynert/substantia innominata (nBM/SI) region of the basal forebrain (BF; highlighted in green). This region contains major cholinergic cell groups that arise from the ventromedial wall of the globus pallidus (GP) and project throughout the cortex (LV: lateral ventricle; IC: internal capsule; CP: caudate putamen). (B) Coronal section depicting the infected BF neurons expressing GFP in the nBM/SI region from an AAV-luc-infused rat. The transduction efficiency of AAV vectors was confirmed by GFP/ChAT immunohistochemistry at the completion of the study. A representative coronal section depicting the GFP-expressing neurons (C; green), ChAT-positive neurons (D; red) and colocalization of GFP and ChAT (E; merged images) from a sample area in the BF is shown. White arrowheads show double-labelled neurons. BF trkA immunoreactivity from representative sections taken from animals infused with AAV-luc (F) and AAV-trkA (G) vector (black arrows point to trkA-positive neurons). (H) Infusion of viral vector expressing trkA shRNA produced profound reduction in trkA immunoreactivity in the nBM/SI region (***, p < 0.001). Besides AAV infusions, animals also received chronic i.c.v infusions of either the vehicle or proNGF antibody (Ab). (I) A representative micrograph of a Nissl-stained section showing the cannula placement (see black arrows for cannula tract) in the right ventricle. (J) Western blots depicting the specificity of proNGF antibody to detect 26 and 32 kDa isoforms of rat proNGF (left). These isoforms were not detected when the antibody was incubated with proNGF blocking peptide (right).

Figure 3

Effects of trkA suppression on attentional performance in the presence and absence of proNGF blockade. All data are Mean ± SEM. (A-C) The index of overall attentional performance expressed as SAT scores. SAT performance was signal duration-dependent but did not differ by block. In general, trkA knockdown rats displayed lower SAT scores for each signal and block, as well as, for the entire session. Bar charts depicting the proportion of hits collapsed by signal-duration (D) and by block (E). Hit rates displayed a similar pattern as SAT scores in terms of the significant main effect of signal. TrkA knockdown aged rats infused with vehicle displayed lower hit rates for each signal duration reflecting that attentional impairments in these animals primarily occurred due to the inability to detect signals. Correct responses on signal trials were not affected by block and displayed no interaction with the manipulation (see section 3.3). (F) Hits averaged over the entire SAT session indicate that sustained proNGF blockade normalized impaired performance on signal trials in trkA knockdown rats. (G) Average correct rejections remained unaffected by either trkA suppression or proNGF blockade but declined when the two manipulations were combined. (H) Response latencies for signal and non-signal trials remained unaffected with any manipulation. (I) Omissions remained relatively low (< 10%) and there were no differences between groups . (main effect of signal: +++ p < 0.001; post hoc comparisons: * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Attentional performance under challenging conditions. All data are Mean ± SEM. Behavioral testing in dSAT involved the presentation of distractors (flashing house light @ 0.5 Hz) in the second block. (A) The presentation of distractors reduced dSAT scores in block 2 as compared to blocks 1 and 3 in all animals. Moreover, dSAT scores remained lower in all blocks in trkA-suppressed rats, and this effect was also emphasized in average dSAT performance (B). (C) The performance in signal trials differed significantly across blocks; hit rates in the distractor and post-distractor blocks declined in all groups. Bar charts depict hit rates for each signal duration (D) and averaged over the entire session (E). dSAT performance in signal trials was signal duration-dependent and this effect interacted with the manipulation. Multiple comparisons indicated that hit rates remained significantly lower for all signal lengths in AAV-trkA + vehicle group. Moreover, proNGF blockade restored this decline in trkA-suppressed rats. However, the correct responses on signals with shorter durations also remained lower in animals with intact trkA receptors but infused with proNGF Ab. Average hit rates exhibited a similar pattern with lower performance in AAV-trkA + vehicle and AAV-luc + proNGF Ab groups, respectively. (F) The performance in non-signal trials dropped for all groups in the distractor block and recovered in the post-distractor block. Correct rejections remained lower throughout the dSAT session in trkA knockdown rats infused with proNGF Ab. These data indicate that lower dSAT scores observed in these animals were mainly attributed to higher false alarms as hit rates were not affected. (LSD: * p < 0.05; ** p < 0.01; *** p < 0.001)

Figure 5

Amperometric recordings of prefrontal cholinergic transmission using choline-sensitive microelectrodes. All data are Mean ± SEM. (A) Representative traces of choline signals evoked by local application of potassium in the medial PFC. These signals reflect ACh release and occur as a consequence of rapid hydrolysis of ACh by acetylcholinesterase. The hydrolyzed choline is oxidized by choline oxidase present on the enzyme at a fixed potential to generate changes in current. B) Cholinergic signal amplitudes were significantly lower in trkA knockdown rats infused with vehicle as compared to trkA intact rats. (LSD: ** p < 0.01).

Figure 6

Cell size of BF cholinergic neurons and cortical cholinergic fiber density. All data are Mean ± SEM. (A) Representative coronal sections depicting ChAT-immunoreactive nBM neurons (marked by white arrowheads) from the sampled areas. The cholinergic soma and dendrites appeared to be shrunken in trkA knockdown rats, though the cross-sectional area did not differ significantly between groups (B). (C-E) ChAT-positive fibers in the medial PFC of control (AAV-luc) and trkA knockdown (AAV-trkA) aged rats that received chronic i.c.v infusions with either vehicle or proNGF Ab. (C) Schematic illustration of the sampling areas for ChAT fiber counts from regions of prelimbic (PrL), cingulate (Cg) and infralimbic (IL) cortex. Counting frames are shown as black squares. (D) Representative photographs illustrate ChAT-immunostained fibers from analyzed prefrontal regions. (E) Bar charts depicting ChAT fiber counts from all treatment groups. The density of cholinergic fibers declined significantly in BF trkA-suppressed rats infused with vehicle as compared to rats with intact trkA receptors in all prefrontal regions. Such a reduction in ChAT fiber density was not observed in AAV-trkA + proNGF animals in both prelimbic and cingulate regions, respectively. However, sustained proNGF blockade per se reduced prefrontal cholinergic processes. (LSD: * , ** p < 0.05, 0.01).