Somatostatin interneurons exhibit enhanced functional output and resilience to axotomy after mild traumatic brain injury

Abstract

Mild traumatic brain injury (mTBI) gives rise to a remarkable breadth of pathobiological consequences, principal among which are traumatic axonal injury and perturbation of the functional integrity of neuronal networks that may arise secondary to the elimination of the presynaptic contribution of axotomized neurons. Because there exists a vast diversity of neocortical neuron subtypes, it is imperative to elucidate the relative vulnerability to axotomy among different subtypes. Toward this end, we exploited SOM-IRES-Cre mice to investigate the consequences of the central fluid percussion model of mTBI on the microanatomical integrity and the functional efficacy of the somatostatin (SOM) interneuron population, one of the principal subtypes of neocortical interneuron. We found that the SOM population is resilient to axotomy, representing only 10% of the global burden of inhibitory interneuron axotomy, a result congruous with past work demonstrating that parvalbumin (PV) interneurons bear most of the burden of interneuron axotomy. However, the intact structure of SOM interneurons after injury did not translate to normal cellular function. One day after mTBI, the SOM population is more intrinsically excitable and demonstrates enhanced synaptic efficacy upon post-synaptic layer 5 pyramidal neurons as measured by optogenetics, yet the global evoked inhibitory tone within layer 5 is stable. Simultaneously, there exists a significant increase in the frequency of miniature inhibitory post-synaptic currents within layer 5 pyramidal neurons. These results are consistent with a scheme in which 1 day after mTBI, SOM interneurons are stimulated to compensate for the release from inhibition of layer 5 pyramidal neurons secondary to the disproportionate axotomy of PV interneurons. The enhancement of SOM interneuron intrinsic excitability and synaptic efficacy may represent the initial phase of a dynamic process of attempted autoregulation of neocortical network homeostasis secondary to mTBI.

Keywords: Axonal injury; Cortical network; Inhibitory interneuron subtypes; Optogenetics; Traumatic brain injury.

Fig. 1.

Limited SOM axotomy following mTBI known to create TAI. Within 3 h post-injury GAD67+ swellings were readily identified throughout layers 2–6 of somatosensory cortex. The vast majority of GAD67+ swellings (A) did not demonstrate expression of tdTomato driven by somatostatin (B). These GAD67+/SS− swellings were sometimes found isolated from the cell body of origin but more often localized to the perisomatic domain. Isolated GAD67+/SS+ swellings were occasionally noted, also occurring either isolated or perisomatically (C-G). Quantitative analysis demonstrated a significant increase in both GAD67+/SS− (t-test, p = 0.021) and GAD67+/SS+ (t-test, p = 0.021) swellings compared to sham (H). Importantly, GAD67+/SS+ swellings constituted only 10% of the total GAD67+ swellings.

Fig. 2.

Intrinsic and membrane properties of SOM neurons are altered after mTBI. A & B. Action potential firing examples at rheobase for control (A) and mTBI (B). C & D. Examples of adaptation that arises during a train of 10 action potentials per 400 msec sweep, for control (C) and TBI (D). Dashed line demonstrates the more hyperpolarized level for the first compared to the last afterhyperpolarization in the train for both control and TBI cells. E. Resting membrane potential (RMP) was significantly more depolarized in the TBI group. F. The time to the first action potential was significantly shorter in the TBI group. G. The slope of the plot of sweep action potential frequency versus injected current was significantly larger in the TBI group. The total adaptation (frequency of first two action potentials/frequency of last two action potentials) for sweeps with 8–10 action potentials was greater in the TBI group. In all cases * = t-test, p < 0.05, for 17 control cells from 6 mice and 24 TBI cells from 4 mice.

Fig. 3.

Responses recorded in fluorescing SOM interneurons in response to activation of ChR2 within SOM interneurons. A. Photomicrographs of example filled neurons. Scale bar is 40 μm. B. Example responses recorded in SOM neurons to ChR2 activation. At durations of 0.6 msec and above, two peaks are apparent (gray lines). Bath application of gabazine abolished the second peak, indicating this component represents a SOM to SOM synaptic response. The remaining peak (black lines) is due to direct ChR2 activation of the SOM interneuron being recorded. C. The direct activation of SOM via ChR2 was not different in TBI compared to control cells. Repeated Measures ANOVA, p > 0.05, n = 13 SOM interneurons from 2 control mice and 12 SOM interneurons from 3 TBI mice. D. The synaptic response was observed in 11 of 13 control and 9 of 12 mTBI SOM interneurons. The peak of the synaptic response was also not significantly different between subject groups. Repeated Measures ANOVA, P > 0.05.

Fig. 4.

Spontaneous inhibitory currents recorded in SOM interneurons. A. All Type I events (events with no overlap with other events) were averaged within each neuron and then averaged across neurons for each subject group (sham in black, TBI in gray), with SEM shown in dashed lines around mean. B. Same as in A, but here the average was normalized to the peak for better visualization of differences in decay timing. There was no significant difference in sIPSC frequency (C), amplitude (D), or rise time (E). The weighted Decay Tau was significantly slower in the TBI group. For these measures a t-test was used, with * = p < 0.05, for 15 neurons from 2 control mice and 26 neurons from 3 TBI mice.

Fig. 5.

Effects of activating ChR2 expressed in SOM interneurons. A, B. Examples of responses of SOM interneurons from control (A) and mTBI (B) animals after long (1) or short (2) pulses of blue light or yellow light (3). C. The number of action potentials produced after varying durations of blue light pulses was similar in control and mTBI groups (repeated measures ANOVA, p > 0.05, n = 9 SOM interneurons from 6 control mice and 7 SOM interneurons from 3 TBI mice). D–F. Recordings from pyramidal neurons during blue light pulses. D. A 400 msec depolarizing pulse produced action potentials within the pyramidal neuron. Yellow light (bottom) had no effect on this action potential train, while blue light (top) aborted the next action potential. E. Blue light pulse given at resting membrane potential in a pyramidal neuron produced an IPSP. F. This non-fluorescing neuron demonstrated an action potential firing pattern typical of pyramidal neurons, in which the first AHP was shorter and more depolarized than the last AHP in the train.

Fig. 6.

ChR2-IPSCs recorded within pyramidal neurons. A, B. Examples of responses to increasing blue light durations applied through the objective above the recorded neuron (2, 10, 30, and 40 msec) in control (A) and mTBI (B). Each waveform is the average of 3 light presentations. Arrow indicates timing of light onset. C. Peak ChR2-IPSC for control (squares, gray line) and mTBI (triangles, black line) across different durations of blue light stimulation. D. Duration of the evoked ChR2-IPSC for same responses as in C. For C and D, repeated measures ANOVA was performed, n = 14 pyramidal neurons from 5 control mice and 11 pyramidal neurons from 4 TBI mice, * = significantly different, p < 0.05.

Fig. 7.

Repetitive ChR2-IPSCs evoked in pyramidal neurons with 2 msec long blue light pulses given at 20 Hz. A, B Five sweep averages show typical responses for control (A) and mTBI (B). Upper scale bar for A and B₁. Lower scale bar for B₂. Starting amplitude for the mTBI was variable, but degree of depression was similar in both cases. C. Peak amplitude of the ChR2-IPSC for control (squares, gray line) and mTBI (triangles, black line), for 13 pyramidal neurons from 5 control mice and 11 pyramidal neurons from 4 TBI mice. D. Synaptic depression shown as the peak response for stimuli 2–5 divided by peak response for stimuli 1. Control and mTBI were not significantly different in the degree of depression (2-way repeated measures ANOVA, p > 0.05).

Fig. 8.

Spontaneous IPSCs recorded in pyramidal neurons. A. Examples of sIPSCs (upper row) and mIPSCs (lower row) recorded in pyramidal neurons from control (left panel) and mTBI (right panel). The frequency of both sIPSCs (B) and mIPSCs (D) was significantly higher in pyramidal neurons of mTBI compared to control cortex. * = p < 0.05, t-tests, n = 26 neurons from 10 control mice and 23 neurons from 9 TBI mice for sIPSCs and 13 neurons from 5 control mice and 9 neurons from 3 TBI mice for mIPSCs. The amplitude of both sIPSCs and mIPSCs was not significantly different between control and mTBI.

Fig. 9.

Electrically-evoked IPSC recorded in pyramidal neurons. Threshold stimulation produced an IPSC on ~50% of trials. Stimulation intensity was then successively increased by doubling the stimulation time. A, B. Shown are typical examples from control (A) and mTBI (B), which are averages of 3 stimulus presentations for 1× – 8×, after which the responses plateaued. C. Peak amplitude of the e-IPSC was not significantly different between control and mTBI groups, n = 24 pyramidal neurons from 4 control mice and 15 pyramidal neurons from 4 TBI mice, 2-way repeated measures ANOVA, p > 0.05.

Fig. 10.

The effect of adding SOM-ChR2 activation to the electrically evoked IPSC. The electrical stimulation was applied as shown in Fig. 9 (gray lines in A-D). In subsequent stimulus applications, blue light was applied immediately after the electrical stimulus for 70 msec (black lines in A-D). Shown are responses to electrical stimulation intensities of 1× and 2× for control (A) and mTBI (B), and for 4× and 8× for control (C) and mTBI (D). E. Control responses. Shown are the peak amplitude for electrical stimulation alone (gray solid line) and for electrical plus light to activate the ChR2 in SOM interneurons (E + L, black dashed line). These were not significantly different, 2-way repeated measures ANOVA, n = 14 paired responses from the same cells. F. Same as E, except for mTBI cortex. A 2-way repeated measures ANOVA showed a significant interaction between condition and stimulus intensity, n = 11. Posthoc analysis showed that the two highest electrical intensities showed a difference between electrical only and electrical + light. G. The peak response in the electrical plus light condition was divided by the peak response in the electrical only condition to generate a percent change for each recorded neuron by stimulus intensity. H. Expansion of the highest 3 electrical stimulating intensities shown in G. A 2-way repeated measures ANOVA showed a significant difference between control and TBI groups, n = 14 pyramidal neurons from 8 control mice and 11 pyramidal neurons from 4 TBI mice, * = p < 0.05.