Nerve growth factor (NGF) augments cortical and hippocampal cholinergic functioning after p75NGF receptor-mediated deafferentation but impairs inhibitory avoidance and induces fear-related behaviors

Abstract

Nerve growth factor (NGF) enhances cholinergic functioning in animals with a compromised cholinergic basal forebrain (CBF). Immunotoxic lesions targeting low-affinity NGF receptor (p75NGF receptor)-bearing CBF neurons provide a selective model for testing the effects of NGF on residual cholinergic neurons. Rats received PBS or the immunotoxin 192IgG-saporin (192Sap) intracerebroventricularly at two doses (1 or 2.7 microg) known to produce different degrees of cholinergic deficit. Seven weeks after lesioning, half of each group received either NGF or cytochrome c intracerebroventricularly for 7 weeks. The two doses of 192Sap produced 50 and 80% depletions of choline acetyltransferase (ChAT) activity in the neocortex and hippocampus. NGF produced the greatest increase in ChAT activity in controls, intermediate in low-lesioned, and smallest in highly lesioned animals. NGF-treated animals showed reduced weight gain, hyper-responsiveness to acoustic stimuli, and decreased inhibitory avoidance. Although general motor behavior was affected by neither 192Sap nor NGF in an open field task, highly lesioned rats took longer to reach the platform during water maze testing. Impaired spatial orientation in finding a hidden platform at the previously acquired position was mitigated by NGF. Hypertrophic changes of residual CBF neurons, Schwann cell hyperplasia, and aberrant axonal sprouting around the medulla were observed in NGF-treated animals only, independent of the preexisting lesion. Our results indicate that NGF has a limited capacity to enhance functioning of residual CBF neurons. More importantly, NGF augmented fear-related behaviors and adverse neuroproliferative changes that may restrict its therapeutic use.

Fig. 1.

Design of the study. Seven weeks after 192Sap-lesioning (pre-treatment), osmotic minipumps were implanted to continuously administer either NGF or CytC. After changing the pump, all animals were tested consecutively using a behavioral battery consisting of open field, startle response, water maze, and passive avoidance. All animals were decapitated at the end of the 7 weeks of treatment for further biochemical and morphological analysis.

Fig. 2.

Loss of p75NGFr-immunoreactive CBF neurons after immunotoxic lesioning accompanied by NGF-induced hypertrophic changes. Representative photomicrographs of the anterior part of the NBM show the different degrees of diminished p75NGFr-immunoreactive neurons after injection of PBS (A), 1 μg of 192Sap (C), and 2.7 μg 192Sap (E). The area of p75NGFr-immunoreactive neurons as well as the density of neuritic sprouting are increased in the PBS/NGF animals (B) but also in the immunotoxic lesioned animals independent of the remaining number of p75NGFr-immunoreactive neurons (D, F).

Fig. 3.

NGF-induced Schwann cell hyperplasia and neuritic sprouting. Representantive photomicrographs of cresyl violet staining of the lower medulla at the level of the hypoglossal nuclei showing the hyperplasia (H) of the Schwann cells beneath the pial cells, the trigeminal tract (TT), and trigeminal nuclei (TN). Note some Schwann cells are present along a vessel perpendicular to the surface of the medulla. A,Low-192Sap/CytC; B, low-192Sap/NGF. All NGF-treated animals, regardless of whether they were lesioned, showed a similar extent of Schwann cell hyperplasia.

Fig. 4.

a, Animal weight—NGF reduces mean body mass. NGF significantly affected the average body weight (F_(5,38) = 14.89; p< 0.0001). NGF-treated animals had a significantly lower mean body mass, independent of the extent of lesioning. Animals were weighed weekly until the end of treatment. Data are expressed as means ± SEM. *Significant difference from the corresponding CytC group; PBS,p < 0.005; low-192Sap, p < 0.0001; high-192Sap, p < 0.0001. b,Animal weight—NGF decreases weight gain. Animals were continuously treated with NGF or CytC via intracerebroventricular cannulas. Body weights were measured week 0 (pump implantation), week 2, week 3.5 (pump change), week 5, and week 7 (end of treatment). NGF significantly altered weight gain throughout the treatment period (time-by-group interaction, F_(20,152) = 15.06;p < 0.0001). In particular, NGF-treated animals never gained body mass similar to the corresponding CytC controls. Eachpoint represents the average body weight of the group during the treatment period. Open squares, PBS/CytC;closed squares, PBS/NGF; open circles, low-192Sap/CytC; closed circles, low-192Sap/NGF;open triangles, high-192Sap/CytC; closed triangles, high-192Sap/NGF.

Fig. 5.

a, Acoustic startle—NGF induces motor hyper-reactivity. NGF treatment resulted in an increased motor reactivity recorded as maximal response to acoustic stimuli (in arbitrary units) averaged across blocks. Note that NGF-induced hyper-reactivity was independent of the preexisting degree of cholinergic deafferentiation. Mean ± SEM displacements are given. *Significant difference from the corresponding CytC-group; PBS, low-192Sap, and high-192Sap, p < 0.005.b, Acoustic startle—habituation to repeated acoustic stimuli is unaffected by NGF and cholinergic deafferentation. Acoustic startle testing consisted of eight blocks of six consecutive trials applying a 115 dB acoustic stimulus. Startle response to the acoustic stimulus is measured by a piezoelectric accelerometer and recorded in arbitrary units. Each point represents the average maximum response of each group. There are significant effects of group (F_(5,38) = 8.1; p< 0.0001) and block (F_(7,266) = 3.5;p < 0.001) but no group-by-block interaction (F_(35,266) = 0.7), indicating that the animals habituated similarly to the repeated acoustic stimuli despite group differences in response. In particular, NGF induced a significantly higher maximal motor response to the consecutive acoustic stimuli compared with the CytC-treated animals. Note that the habituation and the motor reactivity are not affected by the different degrees of cholinergic deafferentation. Open squares, PBS/CytC; closed squares, PBS/NGF; open circles, low-192Sap/CytC; closed circles, low-192Sap/NGF; open triangles, high-192Sap/CytC;closed triangles, high-192Sap/NGF.

Fig. 6.

Water maze—NGF improves spatial orientation during acquisition. Animals were first taught to escape to a marked, submerged platform in the water maze (visible portion of the acquisition). On the following day, the platform was in the same location but unmarked and therefore hidden from the rats' view (hidden portion of the acquisition). Three consecutive two-trial blocks of 90 sec maximum duration were run each day. Overall repeated measures ANOVA indicated a significant group effect (F_(5,38) = 2.5; p< 0.05). The latency to escape to the platform was overall significantly longer for the high-192Sap/CytC, whereas NGF mitigated this deficit (p < 0.05). Latencies diverged by removing the visible cues from the platform. The comparison of the final block of the visible portion (Block 3) to the first block of the hidden portion (Block 4) revealed a significant group effect (F_(5,38) = 4.0; p< 0.006). Only the high-192Sap/CytC group required significantly more time to locate the platform compared with the PBS/CytC (p < 0.005) and the corresponding high-192Sap/NGF groups (p < 0.05). Eachpoint represents the average of the latencies over the two trials within each block. Open squares, PBS/CytC;closed squares, PBS/NGF; open circles, low-192Sap/CytC; closed circles, low-192Sap/NGF;open triangles, high-192Sap/CytC; closed triangles, high-192Sap/NGF.

Fig. 7.

a, IA—spontaneous crossing is unaffected by NGF and cholinergic deafferentation. IA was tested using a two-compartment box. First, each animal was placed in a lighted compartment. A guillotine door to the dark compartment was raised, and a timer began to record step-through latency. Entry was considered complete when all four paws had entered the dark chamber. Single step-through training trial reflects spontaneous crossing from the bright to the dark compartments. Latencies to enter the dark compartment are expressed as means ± SEM. No significant differences were observed among groups (H₍₅₎ = 1.36). Afterward, the door was closed, and a 0.65 mA AC scrambled current was applied.b, IA—NGF impairs 72 hr retention using a single, step-through retention trial. Seventy-two hours after the training trial, single trial, step-through latency as described above was assessed with a maximum time of 600 sec. NGF-treated animals entered the dark compartment significantly earlier compared with the corresponding CytC group. Data are expressed as means ± SEM. *Significant difference from the corresponding CytC-group,p < 0.05, Mann–Whitney Utest.