Matrix metalloproteinase inhibition alters functional and structural correlates of deafferentation-induced sprouting in the dentate gyrus

Abstract

Molecules comprising the extracellular matrix (ECM), and the family of matrix metalloproteinases (MMPs) that regulate them, perform essential functions during neuroplasticity in both developing and adult nervous systems, including substrate guidance during neuritogenesis and the establishment of boundaries for axonal terminal fields. MMP proteolysis of ECM molecules may perform a permissive or inductive role in fiber remodeling and synaptogenesis initiated by deafferentation. This study examined functional and structural effects of MMP inhibition during the early phases of deafferentation-induced sprouting, characterizing components of the degeneration/proliferation cycle that may be dependent on MMP activity. Adult rats received unilateral lesions of the entorhinal cortex to induce collateral sprouting of the crossed temporodentate fiber pathway. This was followed by intraventricular infusion of the MMP inhibitor FN-439 (2.9 mg/kg) or saline vehicle. After 7 d postlesion, rats underwent in vivo electrophysiological recording or histological processing for electron microscopic analysis. Lesioned rats receiving vehicle exhibited normal sprouting and synaptogenesis, with the emergence of the capacity for long-term potentiation (LTP) within the sprouting pathway, and the successful clearance of degenerating terminals with subsequent synaptic proliferation. In contrast, lesioned rats receiving the MMP inhibitor failed to develop the capacity for LTP and showed persistent cellular debris. Current source density analysis also revealed an FN-439-induced disruption of the current sink, normally localized to the middle region of the granule cell dendrites, corresponding to the terminal field of the crossed temporodentate fibers. These results establish a role for MMP-dependent processes in the deafferentation/sprouting cycle.

Figure 1.

Effects of MMP inhibition on LTP in the crossed temporodentate system. A-C, Example field potentials evoked through the crossed perforant path. Pretetanus (top trace) and post-tetanus (bottom trace) waveforms are shown for an unlesioned control rat (A), a lesioned rat given intracerebral ventricular vehicle (B), and a lesioned rat given intracerebral ventricular FN-439 (C). Arrows indicate time of stimulus artifact. D, Group averages of tetanus-induced increases, expressed as percentage of pretetanus baseline, in fEPSP initial slope (left) and in population spike amplitude (right). ^*p < 0.05.

Figure 2.

Effects of MMP inhibition on evoked paired-pulse responses. A, Field potentials evoked with identical paired stimulus pulses, with interpulse interval (50 msec) for a control rat, a lesioned rat given intracerebral ventricular vehicle, and a lesioned rat given intracerebral ventricular FN-439. Note that this stimulation protocol induced a population spike in the second response only for the vehicle-treated lesioned animal. B, Average ratio of the second fEPSP to the first (fEPSP₂/fEPSP₁ × 100), for all experimental groups, using interpulse intervals from 20 to 1000 msec. Note that the predominate pattern for the CTD pathway was a paired-pulse depression, with fEPSP₂ significantly below fEPSP₁ in the following conditions: a, lesioned, FN-439-treated rats (IPI = 40, 50, 100, and 1000 msec); b, unlesioned control rats (IPI = 250 and 500 msec); c, lesioned, vehicle-treated rats (IPI = 500 msec). Also note that for IPI <75 msec, a relative paired-pulse facilitation was observed for the lesioned rats given intracerebral ventricular vehicle. For this group, the fEPSP₂/fEPSP₁ ratio was significantly elevated above that for the control rats (^*p < 0.05) and above that for the lesioned rats given FN-439 (^*p < 0.01).

Figure 3.

CSD analysis of crossed temporodentate fiber system in unlesioned control rat. A, Distance scale along granule cell dendritic axis in dentate molecular layer. B, Field potentials evoked at 25 μm intervals. C, Computed CSD, with positive-going sinks and negative-going sources. D, Shaded CSD (from C) to emphasize major sinks and sources.

Figure 4.

Average CSD results in control group and in lesioned groups with and without MMP inhibition. Curves in each column show the average current sources for each group. Bars at bottom show areas under CSD curves averaged for each rat in each group. Note that the control group and vehicle-treated lesioned group show similar current sink in middle molecular layer, contrasting with a reduced sink in lesioned group given intracerebral ventricular FN-439. Statistical analysis of the averaged CSD showed that one stratum in the FN-439-treated group significantly differed from that in the control group (^*p < 0.01) and the vehicle-treated group (^*p < 0.01).

Figure 5.

The structural effects of acute MMP inhibition with FN-439 on the dentate gyrus molecular layer at 7 d after UEC lesion. In A-C, thick (1 μm) plastic sections stained with toluidine blue show the cytoarchitecture of the dentate molecular layer (brackets) and adjacent granule cell bodies (gcl). In A, the ipsilateral denervated molecular layer of FN-439-treated lesion cases bore clear sites of degenerative debris (bracketed area; example of debris aggregate shown at open arrow), suggestive of the abnormal progress of reactive synaptogenesis. By contrast, the ipsilateral side of the vehicle-treated animals (B) shows little evidence of degenerative debris within the denervated neuropil. Similarly, the molecular layer within contralateral control in FN-439-treated cases exhibited no degenerative pathology (C). At the ultrastructural level (D-F), the dentate molecular layer morphology supports the observations shown in A-C. The effects of acute FN-439 treatment on molecular layer ultrastructure are profound (D). Numerous profiles of degenerative presynaptic terminals (arrows) remain visible. Few intact synapses can be seen, and dendrites appear distended, with disorganized cytoarchitecture and damaged mitochondria (arrowheads). In contrast, the ipsilateral side of cases receiving vehicle (E) exhibit successful synaptic reorganization, with multiple dendritic spines and synaptic profiles visible (arrows). Dendritic processes contain intact cytoskeletal substructure and normal profiles of mitochondria. Notably, the contralateral, nondeafferented neuropil of FN-439-treated animals shows intact synaptic architecture (see arrows in F). Scale bar (in C) A-C, 50 μm; magnification D, E, F, 20,000×.