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. 2019 Oct 1;527(14):2341-2355.
doi: 10.1002/cne.24680. Epub 2019 Mar 22.

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy

Starr Cameron  1 , Ariana Lopez  1   2 , Raisa Glabman  1   3 , Emily Abrams  1 , Shawn Johnson  4 , Cara Field  4 , Frances M D Gulland  4 , Paul S Buckmaster  1   5
Affiliations

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy

Starr Cameron et al. J Comp Neurol. .

Abstract

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin-immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl-stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin-2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2-immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV- or syt2-positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.

Keywords: RRID:AB_10000344; RRID:AB_10013783; dentate gyrus; granule cell; parvalbumin; sea lion; synaptic bouton; synaptotagmin-2; temporal lobe epilepsy.

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Figures

Figure 1
Figure 1
Sclerotic hippocampi were identified by hilar neuron loss in Nissl-stained sections. (Panels a,b) Control sea lion (adult female) with abundant neurons in the hilus (h). g = granule cell layer, m = molecular layer, CA3 = proximal tip of the CA3 pyramidal cell layer. (Panels c,d) Sclerotic hippocampus from a juvenile male with hilar neuron loss but preservation of granule cells. (Panels e,f) Sclerotic hippocampus from an adult female with loss of hilar neurons and granule cells. Scale bar in panel e = 400 μm and applies to panels a,c. Scale bar in panel f = 50 μm and applies to panels b,d.
Figure 2
Figure 2
Nissl-stained coronal sections of cerebral hemispheres reveal unilateral hippocampal sclerosis in an adult female sea lion (panel a). The right hippocampus (arrow) is sclerotic and smaller than the left hippocampus. Scale bar = 1 cm. Higher magnification of the left (panel b) and right hippocampus (panel c) reveals substantial loss of pyramidal cells, hilar neurons, and granule cells in the right hippocampus. h = hilus, g = granule cell layer, m = molecular layer. CA3 = proximal tip of CA3 pyramidal cell layer. Scale bar = 500 μm.
Figure 3
Figure 3
Parvalbumin-immunoreactivity in the left (a) and right (b) dentate gyrus of an adult female sea lion with right unilateral hippocampal sclerosis. Cell layers indicated. h = hilus, g = granule cell layer, m = molecular layer, CA3 = proximal tip of CA3 pyramidal cell layer. Scale bar = 500 μm.
Figure 4
Figure 4
Parvalbumin-immunoreactivity (panels a-c), synaptotagmin-2-immunoreactivity (panels d-f), and Nissl staining (panels g-i) of hippocampi from an adult female control sea lion (panels a,d,g), a juvenile male sea lion with bilateral sclerosis (panels b,e,h), and an adult male sea lion with bilateral sclerosis (panels c,f,i). (g) Cell layers indicated. h = hilus, g = granule cell layer, m = molecular layer, CA3 = proximal tip of the CA3 pyramidal cell layer. In one of the sea lions with bilaterally sclerotic hippocampi, arrows indicate part of the granule cell layer containing granule cells (panel h) but devoid of parvalbumin- (panel b) and synaptotagmin-2-immunoreactive boutons (panel e). (Panel f) In another sea lion with bilaterally sclerotic hippocampi, arrowheads indicate aberrant synaptotagmin-2-immunoreactivity in the inner molecular layer and hilus. Scale bar = 500 μm.
Figure 5
Figure 5
Higher magnification views of parvalbumin-immunoreactivity in the dentate gyrus of the same control sea lion (panels a,b) and same two sea lions with bilateral sclerosis (panels c,d and e,f) shown at lower magnification in Figure 4. A fusiform (arrow) and a multipolar (double arrow) soma are indicated (panels a and b). Some boutons are indicated by arrowheads (panels b,d,f). h = hilus, g = granule cell layer, m = molecular layer. Scale bar in panel e = 100 μm and applies to panels a,c. Scale bar in panel f = 20 μm and applies to panels b,d.
Figure 6
Figure 6
Number of granule cells (panel a), parvalbumin-immunoreactive neurons (panel b), granule cells per parvalbumin neuron (panel c), parvalbumin-immunoreactive boutons (panel d), parvalbumin boutons per parvalbumin neuron (panel e), and parvalbumin boutons per granule cell (panel f) in the dentate gyrus of control sea lions, sea lions with bilaterally sclerotic hippocampi, and sea lions with unilaterally sclerotic hippocampi (divided into non-sclerotic and sclerotic groups). Markers indicate values from individual sea lions. Bars indicate averages. Asterisks indicate significantly different from the control group, p < 0.05, Kruskal-Wallis ANOVA on ranks with Dunn’s method. ‡ indicates groups in which one outlier value (≥ 2.5-times from the average) is higher than the y-axis scale of the plot.
Figure 7
Figure 7
Higher magnification views of synaptotagmin-2-immunoreactivity in the dentate gyrus of the same control sea lion (panels a,b) and same two sea lions with bilateral sclerosis (panels c,d and e,f) shown at lower magnification in Figure 4. Some boutons are indicated by arrowheads (panels b,d,f). h = hilus, g = granule cell layer, iml = inner molecular layer, mml = middle molecular layer. Scale bar in panel e = 100 μm and applies to panels a,c. Scale bar in panel f = 20 μm and applies to panels b,d.
Figure 8
Figure 8
Number of synaptotagmin-2-immunoreactive boutons (panel a), synaptotagmin-2 boutons per parvalbumin bouton (panel b), and synaptotagmin-2 boutons per granule cell (panel c) in the dentate gyrus of control sea lions, sea lions with bilaterally sclerotic hippocampi, and sea lions with unilaterally sclerotic hippocampi (divided into non-sclerotic and sclerotic groups). Markers indicate values from individual sea lions. Bars indicate averages. Asterisks indicate significantly different from the control group, p < 0.05, Kruskal-Wallis ANOVA on ranks with Dunn’s method. ‡ indicates groups in which one outlier value (> 3.5-times from the average) is higher than the y-axis scale of the plot.
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