Instantaneous perturbation of dentate interneuronal networks by a pressure wave-transient delivered to the neocortex

Abstract

Whole-cell patch-clamp recordings and immunocytochemical experiments were performed to determine the short- and long-term effects of lateral fluid percussion head injury on the perisomatic inhibitory control of dentate granule cells in the adult rat, with special reference to the development of trauma-induced hyperexcitability. One week after the delivery of a single, moderate (2.0-2.2 atm) mechanical pressure wave to the neocortex, the feed-forward inhibitory control of dentate granule cell discharges was compromised, and the frequency of miniature IPSCs was decreased. Consistent with the electrophysiological data, the number of hilar parvalbumin (PV)- and cholecystokinin (CCK)-positive dentate interneurons supplying the inhibitory innervation of the perisomatic region of granule cells was decreased weeks and months after head injury. The initial injury to the hilar neurons took place instantaneously after the impact and did not require the recruitment of active physiological processes. Furthermore, the decrease in the number of PV- and CCK-positive hilar interneurons was similar to the decrease in the number of the AMPA-type glutamate receptor subunit 2/3-immunoreactive mossy cells, indicating that the pressure wave-transient causes injurious physical stretching and bending of most cells that are large and not tightly packed in a cell layer. These results reveal for the first time that moderate pressure wave-transients, triggered by traumatic head injury episodes, impact the dentate neuronal network in a unique temporal and spatial pattern, resulting in a net decrease in the perisomatic control of granule cell discharges.

Fig. 1.

Decreased feed-forward inhibition of dentate granule cells 1 week after fluid percussion head injury.A, Field recordings of perforant path-evoked granule cell responses 1 week after moderate head injury in slices obtained from a fluid percussion-injured (FPI) and an age-matched, sham-operated control animal, at three intensities of stimulation (low, 500 μA; medium, 2 mA; high, 6 mA). The responses recorded in the slice from the fluid percussion-injured animal show population discharge, indicating a decreased ability of the interneuronal network to control the feed-forward activation of granule cells. Note that the EPSPs at lower intensities of stimulation were not statistically different. B, In the presence of the GABA_A receptor antagonist bicuculline (BMI), population spikes can be observed in control slices, indicating that the absence of population spikes in control ACSF in control slices is attributable to the powerful feed-forward activation of interneurons inhibiting granule cell discharges via GABA_A receptors. C, Summary of data obtained in control medium (as shown in A). Note that the amplitude of the population spike in the slices from fluid percussion-injured animals was larger than in control.

Fig. 2.

Decreased frequency of mIPSCs in dentate granule cells after head injury. A, The traces are representative recordings of mIPSCs (at −60 mV with Cl⁻-filled patch pipettes) from dentate granule cells of age-matched, sham-operated control and fluid percussion-injured (FPI) animals, 1 week after injury. B, The bar graphs indicate a significantly increased inter-event interval (a decreased frequency) of the mIPSCs from the injured animals. C, The figure shows an example of average mIPSCs recorded from granule cells of control and fluid percussion-injured animals; the average currents (○) showed complete overlap. The currents were accurately described (– – –) by the sum of a single exponential rise and a single exponential decay. The amplitude and kinetics of the mIPSCs were not significantly different after fluid percussion injury (for the numerical values of the amplitude and kinetics data, see text).

Fig. 3.

The number of PV-immunostained neurons in the hilus and the density of PV-positive fibers in the hilus and in the granule cell layer are decreased after head injury. A,B, PV immunostaining in age-matched, sham-operated control. Note the presence of PV-positive cells and processes in the hilus and in and above the granule cell layer. The area outlined in A is shown at higher magnification in B. C, D, One week after head injury, the hilus contains fewer PV-positive cells and processes. Similarly, there is a considerable decrease in the density of PV-positive fibers in and above the granule cell layer (e.g., compare B andD). However, despite the great decrease in the number of hilar PV-positive cells, numerous PV-immunostained neurons can be observed in the granule cell layer. The area outlined inC is shown at higher magnification in D. Magnifications: A, C, 112×; B, D, 500×.H, Hilus; GCL, granule cell layer.

Fig. 4.

Head injury-induced alterations in the interneuronal circuitry of the dentate gyrus. A, Fluid percussion injury causes a significant decrease in the number of parvalbumin (PV)-immunostained cells in the hilus, without a change in the number of PV-positive cells in the granule cell and molecular layers, 1 week after impact, compared with age-matched, sham-operated controls. B, Percentage decrease in the number of PV-, CCK-, and GluR2/3-positive cells in the hilus of the dentate gyrus (in each case, with respect to age-matched, sham-operated controls). Note that the post-traumatic percentage decrease in the number of PV- and CCK-positive cells in the hilus is similar to the decrease in the number of GluR2/3-immunostained mossy cells (one of the most injury-sensitive cell types in the entire brain in various models of epilepsy and ischemia), as well as to the average decrease in the total number of hilar cells (as determined from Nissl-stained sections).

Fig. 5.

Decrease in the number of the GluR2/3-immunoreactive mossy hilar cells after fluid percussion injury.A, GluR2/3 immunostaining shows numerous mossy cells (Leranth et al., 1996) in the hilus of the dentate gyrus of an age-matched, sham-operated control animal. B, The number of GluR2/3-immunostained cells in the hilus is decreased 1 week after head injury. Magnification: 90×. H, Hilus;GCL, granule cell layer.

Fig. 6.

The trauma-induced perturbation of the dentate interneuronal network is immediate and does not require the recruitment of active physiological processes. A, C, Silver staining in an animal that was transcardially fixed immediately after fluid percussion injury. Note the presence of numerous darkly stained cells in the dentate hilus and also a subset of the darkly stained cells (interneurons, see below) in the granule cell layer. Note the lack of staining of CA1 neurons (in A) and granule cells (e.g., in C). B, Sections from age-matched, sham-operated control animals remained unstained. C, D, The cells that appeared darkly stained in the granule cell layer after fluid percussion injury had large and most often pyramidal-shaped somata, indicating that these cells were basket cells. E, F, The pattern of silver-stained cells in the hilus and the granule cell layer was essentially unchanged when fluid percussion injury was performed in animals that were prefixed with a fixative solution, indicating that the initial mechanical injury to dentate interneurons does not require the active recruitment of physiological processes. Magnifications: A–C, 125×;D, 500×; E, F, 190×; F, 430×. H, Hilus; GCL, granule cell layer;F, fissure.

Fig. 7.

SPR immunostaining is severely decreased in the hilus, but the number of SPR-immunostained cells remains unchanged in the granule cell layer. A, SPR immunostaining shows a dense meshwork of GABAergic cells (curved arrows) and processes (Acsády et al., 1997) in the dentate hilus in a section from an age-matched, sham-operated control, and the presence of pyramidal-shaped cells (straight arrows) in the granule cell layer. B, After fluid percussion injury, the density of SPR-positive cells and processes is greatly decreased in the hilus, indicating a disturbance of the GABAergic hilar network. However, the pyramidal-shaped interneurons can still be observed in the granule cell layer (arrows). Magnification: 450×.H, Hilus; GCL, granule cell layer.