Concussive Head Trauma Deranges Axon Initial Segment Function in Axotomized and Intact Layer 5 Pyramidal Neurons.

The axon initial segment (AIS) is a critical locus of control of action potential (AP) generation and neuronal information synthesis. Concussive traumatic brain injury gives rise to diffuse axotomy, and the majority of neocortical axonal injury arises at the AIS. Consequently, concussive traumatic brain injury might profoundly disrupt the functional specialization of this region. To investigate this hypothesis, one and two days after mild central fluid percussion injury in Thy1-YFP-H mice, we recorded high-resolution APs from axotomized and adjacent intact layer 5 pyramidal neurons and applied a second derivative (2o) analysis to measure the AIS- and soma-regional contributions to the AP upstroke. All layer 5 pyramidal neurons recorded from sham animals manifested two stark 2o peaks separated by a negative intervening slope. In contrast, within injured mice, we discovered a subset of axotomized layer 5 pyramidal neurons in which the AIS-regional 2o peak was abolished, a functional perturbation associated with diminished excitability, axonal sprouting and distention of the AIS as assessed by staining for ankyrin-G. Our analysis revealed an additional subpopulation of both axotomized and intact layer 5 pyramidal neurons that manifested a melding together of the AIS- and soma-regional 2o peaks, suggesting a more subtle aberration of sodium channel function and/or translocation of the AIS initiation zone closer to the soma. When these experiments were repeated in animals in which cyclophilin-D was knocked out, these effects were ameliorated, suggesting that trauma-induced AIS functional perturbation is associated with mitochondrial calcium dysregulation.

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

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.

Enhanced responses to somatostatin interneuron activation in developmentally malformed cortex.

Intractable epilepsy is commonly associated with developmental cortical malformations. Using the rodent freeze lesion model, we have sought the underlying circuit abnormalities contributing to the epileptiform activity that occurs in association with the structural pathology of four-layered microgyria. We showed previously that within the epileptogenic paramicrogyral region (PMR) surrounding the malformation, non-fast-spiking neurons commonly containing somatostatin (SSt) exhibit alterations, including having a greater maximum firing rate. Here we examined the output of SSt interneurons with optogenetics, using SSt-Cre mice mated to mice with floxed channelrhodopsin-2. Voltage clamp recordings from layer V pyramidal neurons in ex vivo slices had significantly enhanced SSt-evoked inhibitory postsynaptic currents in PMR cortex compared to control. In addition, under conditions of low-Mg2+ artificial cerebral spinal fluid (aCSF), light activation of the SSt neurons evoked field potential epileptiform activity in the PMR cortex, but not in control. These data suggest that within the PMR cortex, SSts have a significantly larger effect on excitatory neurons. Surprisingly, the network effect of this enhanced inhibition is hyperexcitability with propagating epileptiform activity, perhaps due to disinhibition of other interneuron cell types or to enhanced synchrony of excitatory cortical elements. This identification creates a new locus for potential modulation of epileptiform activity associated with cortical malformation.

Mild Traumatic Brain Injury Induces Structural and Functional Disconnection of Local Neocortical Inhibitory Networks via Parvalbumin Interneuron Diffuse Axonal Injury.

Diffuse axonal injury (DAI) plays a major role in cortical network dysfunction posited to cause excitatory/inhibitory imbalance after mild traumatic brain injury (mTBI). Current thought holds that white matter (WM) is uniquely vulnerable to DAI. However, clinically diagnosed mTBI is not always associated with WM DAI. This suggests an undetected neocortical pathophysiology, implicating GABAergic interneurons. To evaluate this possibility, we used mild central fluid percussion injury to generate DAI in mice with Cre-driven tdTomato labeling of parvalbumin (PV) interneurons. We followed tdTomato+ profiles using confocal and electron microscopy, together with patch-clamp analysis to probe for DAI-mediated neocortical GABAergic interneuron disruption. Within 3 h post-mTBI tdTomato+ perisomatic axonal injury (PSAI) was found across somatosensory layers 2-6. The DAI marker amyloid precursor protein colocalized with GAD67 immunoreactivity within tdTomato+ PSAI, representing the majority of GABAergic interneuron DAI. At 24 h post-mTBI, we used phospho-c-Jun, a surrogate DAI marker, for retrograde assessments of sustaining somas. Via this approach, we estimated DAI occurs in ~9% of total tdTomato+ interneurons, representing ~14% of pan-neuronal DAI. Patch-clamp recordings of tdTomato+ interneurons revealed decreased inhibitory transmission. Overall, these data show that PV interneuron DAI is a consistent and significant feature of experimental mTBI with important implications for cortical network dysfunction.

Inflammatory demyelination alters subcortical visual circuits.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease classically associated with axonal damage and loss; more recently, however, synaptic changes have been recognized as additional contributing factors. An anatomical area commonly affected in MS is the visual pathway; yet, changes other than those associated with inflammatory demyelination of the optic nerve, i.e., optic neuritis, have not been described in detail. METHODS: Adult mice were subjected to a diet containing cuprizone to mimic certain aspects of inflammatory demyelination as seen in MS. Demyelination and inflammation were assessed by real-time polymerase chain reaction and immunohistochemistry. Synaptic changes associated with inflammatory demyelination in the dorsal lateral geniculate nucleus (dLGN) were determined by immunohistochemistry, Western blot analysis, and electrophysiological field potential recordings. RESULTS: In the cuprizone model, demyelination was observed in retinorecipient regions of the subcortical visual system, in particular the dLGN, where it was found accompanied by microglia activation and astrogliosis. In contrast, anterior parts of the pathway, i.e., the optic nerve and tract, appeared largely unaffected. Under the inflammatory demyelinating conditions, as seen in the dLGN of cuprizone-treated mice, there was an overall decrease in excitatory synaptic inputs from retinal ganglion cells. At the same time, the number of synaptic complexes arising from gamma-aminobutyric acid (GABA)-generating inhibitory neurons was found increased, as were the synapses that contain the N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B and converge onto inhibitory neurons. These synaptic changes were functionally found associated with a shift toward an overall increase in network inhibition. CONCLUSIONS: Using the cuprizone model of inflammatory demyelination, our data reveal a novel form of synaptic (mal)adaption in the CNS that is characterized by a shift of the excitation/inhibition balance toward inhibitory network activity associated with an increase in GABAergic inhibitory synapses and a possible increase in excitatory input onto inhibitory interneurons. In addition, our data recognize the cuprizone model as a suitable tool in which to assess the effects of inflammatory demyelination on subcortical retinorecipient regions of the visual system, such as the dLGN, in the absence of overt optic neuritis.

Mild Traumatic Brain Injury Evokes Pyramidal Neuron Axon Initial Segment Plasticity and Diffuse Presynaptic Inhibitory Terminal Loss.

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output.

Knockout of Cyclophilin-D Provides Partial Amelioration of Intrinsic and Synaptic Properties Altered by Mild Traumatic Brain Injury.

Mitochondria are central to cell survival and Ca(2+) homeostasis due to their intracellular buffering capabilities. Mitochondrial dysfunction resulting in mitochondrial permeability transition pore (mPTP) opening has been reported after mild traumatic brain injury (mTBI). Cyclosporine A provides protection against the mPTP opening through its interaction with cyclophilin-D (CypD). A recent study has found that the extent of axonal injury after mTBI was diminished in neocortex in cyclophilin-D knockout (CypDKO) mice. Here we tested whether this CypDKO could also provide protection from the increased intrinsic and synaptic neuronal excitability previously described after mTBI in a mild central fluid percussion injury mice model. CypDKO mice were crossed with mice expressing yellow fluorescent protein (YFP) in layer V pyramidal neurons in neocortex to create CypDKO/YFP-H mice. Whole cell patch clamp recordings from axotomized (AX) and intact (IN) YFP+ layer V pyramidal neurons were made 1 and 2 days after sham or mTBI in slices from CypDKO/YFP-H mice. Both excitatory post synaptic currents (EPSCs) recorded in voltage clamp and intrinsic cellular properties, including action potential (AP), afterhyperpolarization (AHP), and depolarizing after potential (DAP) characteristics recorded in current clamp were evaluated. There was no significant difference between sham and mTBI for either spontaneous or miniature EPSC frequency, suggesting that CypDKO ameliorates excitatory synaptic abnormalities. There was a partial amelioration of intrinsic properties altered by mTBI. Alleviated were the increased slope of the AP frequency vs. injected current plot, the increased AP, AHP and DAP amplitudes. Other properties that saw a reversal that became significant in the opposite direction include the current rheobase and AP overshoot. The AP threshold remained depolarized and the input resistance remained increased in mTBI compared to sham. Additional altered properties suggest that the CypDKO likely has a direct effect on membrane properties, rather than producing a selective reduction of the effects of mTBI. These results suggest that inhibiting CypD after TBI is an effective strategy to reduce synaptic hyperexcitation, making it a continued target for potential treatment of network abnormalities.

Increased Network Excitability Due to Altered Synaptic Inputs to Neocortical Layer V Intact and Axotomized Pyramidal Neurons after Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) can produce long lasting cognitive dysfunction. There is typically no cell death and only diffuse structural injury after mTBI. Thus, functional changes in intact neurons may contribute to symptoms. We have previously shown altered intrinsic properties of axotomized and intact neurons within 2 d after a central fluid percussion injury in mice expressing yellow fluorescent protein (YFP) that allow identification of axonal state prior to recording. Here, whole-cell patch clamp recordings were used to examine synaptic properties of YFP(+) layer V pyramidal neurons. An increased frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was recorded from axotomized neurons at 1 d and intact neurons at 2 d after injury, likely reflecting an increased number of afferents. This also was reflected in the increased amplitude of the EPSC evoked by local extracellular stimulation for all neurons from injured cortex and increased likelihood of producing an action potential for intact cells. Field potentials recorded in superficial layers after online deep layer stimulation contained a single negative peak in controls but multiple negative peaks in injured tissue. The amplitude of this evoked negativity was significantly larger than controls over a series of stimulus intensities at both the 1 d and 2 d survival times. Interictal-like spikes never occurred in the field potential recordings from controls but were observed in 20-80% of stimulus presentations in injured cortex. Together, these results suggest an overall increase in network excitability and the production of particularly powerful (intact) neurons that have both increased intrinsic and synaptic excitability.

Simulating vertical and horizontal inhibition with short-term dynamics in a multi-column multi-layer model of neocortex.

The paper introduces a multi-layer multi-column model of the cortex that uses four different neuron types and short-term plasticity dynamics. It was designed with details of neuronal connectivity available in the literature and meets these conditions: (1) biologically accurate laminar and columnar flows of activity, (2) normal function of low-threshold spiking and fast spiking neurons, and (3) ability to generate different stages of epileptiform activity. With these characteristics the model allows for modeling lesioned or malformed cortex, i.e. examine properties of developmentally malformed cortex in which the balance between inhibitory neuron subtypes is disturbed.

Early susceptibility for epileptiform activity in malformed cortex.

Despite early disruption of developmental processes, hyperexcitability is often delayed after the induction of cortical malformations. In the freeze-lesion model of microgyria, interictal activity cannot be evoked in vitro until postnatal day (P)12, despite the increased excitatory afferent input to the epileptogenic region by P10. In order to determine the most critical time period for assessment of epileptogenic mechanisms, here we have used low-Mg(2+) aCSF as a second hit after the neonatal freeze lesion to examine whether there is an increased susceptibility prior to the overt expression of epileptiform activity. This two-hit model produced increased interictal activity in freeze-lesioned relative to control cortex. We quantified this with measures of incidence by sweep, time to first epileptiform event, and magnitude of late activity. The increase was present even in the P7-9 survival group, before increased excitatory afferents invade, as well as in the P10-11 and P12-15 groups. In our young adult group (P28-36), the amount of interictal activity did not differ, but only the lesioned cortices produced ictal activity. We conclude that epileptogenic processes begin early and continue beyond the expression of interictal activity, with different time courses for susceptibility for interictal and ictal activity.

Biological Restraint on the Izhikevich Neuron Model Essential for Seizure Modeling.

We propose a simple modification of the Izhikevich neuron model to restrict firing rates of neurons. We demonstrate how this modification affects overall network activity using a simple artificial network. Such restraint on the Izhikevich neuron model would be especially important in larger scale simulations or when frequency dependent short-term plasticity is one of the network components. Although maximum firing rates are most likely exceeded in simulations of seizure like activity or other conditions that promote excessive excitation, we show that restriction of neuronal firing frequencies has impact even on small networks with moderate levels of input.

Simulating lesions in multi-layer, multi-columnar model of neocortex.

The paper presents results of modeling global and focal loss of layers in a multi-columnar model of neocortex. Specifically, the spread of activity across columns in conditions of inhibitory blockade is compared. With very low inhibition activity spreads through all layers, however, deep layers are critical for spread of activity when inhibition is only moderately blocked.

Electrophysiological abnormalities in both axotomized and nonaxotomized pyramidal neurons following mild traumatic brain injury.

Mild traumatic brain injury (mTBI) often produces lasting detrimental effects on cognitive processes. The mechanisms underlying neurological abnormalities have not been fully identified, in part due to the diffuse pathology underlying mTBI. Here we employ a mouse model of mTBI that allows for identification of both axotomized and intact neurons in the living cortical slice via neuronal expression of yellow fluorescent protein. Both axotomized and intact neurons recorded within injured cortex are healthy with a normal resting membrane potential, time constant (τ), and input resistance (R(in)). In control cortex, 25% of cells show an intrinsically bursting action potential (AP) firing pattern, and the rest respond to injected depolarizing current with a regular-spiking pattern. At 2 d postinjury, intrinsic bursting activity is lost within the intact population. The AP amplitude is increased and afterhyperpolarization duration decreased in axotomized neurons at 1 and 2 d postinjury. In contrast, intact neurons also show these changes at 1 d, but recover by 2 d postinjury. Two measures suggest an initial decrease in excitability in axotomized neurons followed by an increase in excitability within intact neurons. The rheobase is significantly increased in axotomized neurons at 1 d postinjury. The slope of the plot of AP frequency versus injected current is larger for intact neurons at 2 d postinjury. Together, these results demonstrate that intact and axotomized neurons are both affected by mTBI, resulting in different changes in neuronal excitability that may contribute to network dysfunction following TBI.

Synaptic information transfer in computer models of neocortical columns.

Understanding the direction and quantity of information flowing in neuronal networks is a fundamental problem in neuroscience. Brains and neuronal networks must at the same time store information about the world and react to information in the world. We sought to measure how the activity of the network alters information flow from inputs to output patterns. Using neocortical column neuronal network simulations, we demonstrated that networks with greater internal connectivity reduced input/output correlations from excitatory synapses and decreased negative correlations from inhibitory synapses, measured by Kendall's τ correlation. Both of these changes were associated with reduction in information flow, measured by normalized transfer entropy (nTE). Information handling by the network reflected the degree of internal connectivity. With no internal connectivity, the feedforward network transformed inputs through nonlinear summation and thresholding. With greater connectivity strength, the recurrent network translated activity and information due to contribution of activity from intrinsic network dynamics. This dynamic contribution amounts to added information drawn from that stored in the network. At still higher internal synaptic strength, the network corrupted the external information, producing a state where little external information came through. The association of increased information retrieved from the network with increased gamma power supports the notion of gamma oscillations playing a role in information processing.

Altered intrinsic properties of neuronal subtypes in malformed epileptogenic cortex.

Neuronal intrinsic properties control action potential firing rates and serve to define particular neuronal subtypes. Changes in intrinsic properties have previously been shown to contribute to hyperexcitability in a number of epilepsy models. Here we examined whether a developmental insult producing the cortical malformation of microgyria altered the identity or firing properties of layer V pyramidal neurons and two interneuron subtypes. Trains of action potentials were elicited with a series of current injection steps during whole cell patch clamp recordings. Cells in malformed cortex identified as having an apical dendrite had firing patterns similar to control pyramidal neurons. The duration of the second action potential in the train was increased in paramicrogyral (PMG) pyramidal cells, suggesting that these cells may be in an immature state, as was previously found for layer II/III pyramidal neurons. Based on stereotypical firing patterns and other intrinsic properties, fast-spiking (FS) and low threshold-spiking (LTS) interneuron subpopulations were clearly identified in both control and malformed cortex. Most intrinsic properties measured in malformed cortex were unchanged, suggesting that subtype identity is maintained. However, LTS interneurons in lesioned cortex had increased maximum firing frequency, decreased initial afterhyperpolarization duration, and increased total adaptation ratio compared to control LTS cells. FS interneurons demonstrated decreased maximum firing frequencies in malformed cortex compared to control FS cells. These changes may increase the efficacy of LTS while decreasing the effectiveness of FS interneurons. These data indicate that differential alterations of individual neuronal subpopulations may endow them with specific characteristics that promote epileptogenesis.