Robust perisomatic GABAergic self-innervation inhibits basket cells in the human and mouse supragranular neocortex.

Inhibitory autapses are self-innervating synaptic connections in GABAergic interneurons in the brain. Autapses in neocortical layers have not been systematically investigated, and their function in different mammalian species and specific interneuron types is poorly known. We investigated GABAergic parvalbumin-expressing basket cells (pvBCs) in layer 2/3 (L2/3) in human neocortical tissue resected in deep-brain surgery, and in mice as control. Most pvBCs showed robust GABAAR-mediated self-innervation in both species, but autapses were rare in nonfast-spiking GABAergic interneurons. Light- and electron microscopy analyses revealed pvBC axons innervating their own soma and proximal dendrites. GABAergic self-inhibition conductance was similar in human and mouse pvBCs and comparable to that of synapses from pvBCs to other L2/3 neurons. Autaptic conductance prolonged somatic inhibition in pvBCs after a spike and inhibited repetitive firing. Perisomatic autaptic inhibition is common in both human and mouse pvBCs of supragranular neocortex, where they efficiently control discharge of the pvBCs.

Neuregulin 1 Type I Overexpression Is Associated with Reduced NMDA Receptor-Mediated Synaptic Signaling in Hippocampal Interneurons Expressing PV or CCK.

Hypofunction of N-methyl-d-aspartate receptors (NMDARs) in inhibitory GABAergic interneurons is implicated in the pathophysiology of schizophrenia (SZ), a heritable disorder with many susceptibility genes. However, it is still unclear how SZ risk genes interfere with NMDAR-mediated synaptic transmission in diverse inhibitory interneuron populations. One putative risk gene is neuregulin 1 (NRG1), which signals via the receptor tyrosine kinase ErbB4, itself a schizophrenia risk gene. The type I isoform of NRG1 shows increased expression in the brain of SZ patients, and ErbB4 is enriched in GABAergic interneurons expressing parvalbumin (PV) or cholecystokinin (CCK). Here, we investigated ErbB4 expression and synaptic transmission in interneuronal populations of the hippocampus of transgenic mice overexpressing NRG1 type I (NRG1tg-type-I mice). Immunohistochemical analyses confirmed that ErbB4 was coexpressed with either PV or CCK in hippocampal interneurons, but we observed a reduced number of ErbB4-immunopositive interneurons in the NRG1tg-type-I mice. NMDAR-mediated currents in interneurons expressing PV (including PV+ basket cells) or CCK were reduced in NRG1tg-type-I mice compared to their littermate controls. We found no difference in AMPA receptor-mediated currents. Optogenetic activation (5 pulses at 20 Hz) of local glutamatergic fibers revealed a decreased NMDAR-mediated contribution to disynaptic GABAergic inhibition of pyramidal cells in the NRG1tg-type-I mice. GABAergic synaptic transmission from either PV+ or CCK+ interneurons, and glutamatergic transmission onto pyramidal cells, did not significantly differ between genotypes. The results indicate that synaptic NMDAR-mediated signaling in hippocampal interneurons is sensitive to chronically elevated NGR1 type I levels. This may contribute to the pathophysiological consequences of increased NRG1 expression in SZ.

High-Precision Fast-Spiking Basket Cell Discharges during Complex Events in the Human Neocortex.

In the human neocortex, solitary action potentials in some layer 2-3 pyramidal cells (PCs) trigger brief episodes of network activity known as complex events through strong excitatory synapses that specifically innervate GABAergic interneurons. Yet, how these "master PCs" configure the local network activity is not well understood. We report that single spikes in the PCs, studied here in synaptically connected cell pairs in frontal or temporal neocortical areas of both males and females, elicit firing of fast-spiking basket cells (FSBCs) with a short delay (on average 2.7 ms). The FSBC discharge is triggered by 13 mV (on average) monosynaptic EPSPs, and the action potential is time locked to the master PC spike with high temporal precision, showing little jitter in delay. In the complex events, the FSBC discharge occurs in the beginning of the activity episode, forming the first wave of the complex event activity. Firing of FSBCs generates GABAergic IPSCs with fast kinetics in layer 2-3 PCs, and similar IPSCs regularly occur time locked to master PC spikes in the beginning of the complex events with high probability and short (median 4.1 ms) delay with little jitter. In comparison, discharge of nonfast spiking interneurons (non-FSINs) investigated here appears inconsistently in the complex events and shows low probability. Thus, firing of layer 2-3 FSBCs with high temporal fidelity characterizes early phase of the complex events in the human neocortex.

Presymptomatically applied AMPA receptor antagonist prevents calcium increase in vulnerable type of motor axon terminals of mice modeling amyotrophic lateral sclerosis.

Increased intracellular calcium (Ca), which might be the consequence of an excess influx through Ca-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, plays a crucial role in degeneration of motor neurons. Previously we demonstrated that the presymptomatic application of AMPA receptor antagonist, talampanel, could reduce Ca elevation in spinal motor neurons of mice carrying the G93A mutation of superoxide dismutase 1 (SOD1), modeling amyotrophic lateral sclerosis (ALS). It remained to be examined whether the remote, functionally semi-autonomous motor axon terminals could be rescued from the Ca overload, or if the terminals, where the degeneration possibly starts, already experience intractable changes at early time points. Thus using electron microscopic techniques, we measured the Ca level of motor axon terminals in the interosseus muscle of the SOD1 mutant animals, which are prototypes of vulnerable nerve endings in ALS. In line with the results obtained in the perikarya, talampanel treatment could reduce Ca increase evoked by the presence of mutant SOD1 in the axon terminals if the treatment was started presymptomatically but not at an early symptomatic stage. We also tested the Ca level in the cell bodies and axon terminals of the oculomotor neurons, which are resistant to the disease. Neither Ca increase, nor talampanel effect could be demonstrated at either time point. This is consistent with the observations that oculomotor neurons contain increased level of Ca buffer, which could reduce excess Ca load, and they also express glutamate receptor subunit type 2, which renders AMPA receptors impermeable to Ca.

Axotomy Leads to Reduced Calcium Increase and Earlier Termination of CCL2 Release in Spinal Motoneurons with Upregulated Parvalbumin Followed by Decreased Neighboring Microglial Activation.

BACKGROUND: Motoneurons with naturally elevated calcium binding protein content, such as parvalbumin, are more resistant against injury. Furthermore, increase of intracellular calcium, which plays a pivotal role in injury of neurons, could be moderated by elevating their calcium binding proteins. OBJECTIVE: To test whether by elevating parvalbumin content of motoneurons, activation of neighboring microglial cells, a robust component of the inflammatory reaction after injury, could be influenced. METHODS: Mice overexpressing neuronal parvalbumin were derived and the spinal motoneurons were challenged by cutting the sciatic nerve. At postoperative days 1, 4, 7, 14 and 21 the change of the chemokine ligand 2 immunostaining in the motoneurons and the activation of microglial cells, measured as alterations in CD11b immunostaining were determined. Calcium level of motoneurons was tested electron microscopically at postoperative day 7. RESULTS: After axotomy, increased level of chemokine ligand 2 was detected in the lumbar motoneurons. The staining intensity reached its maximum at day 7 and decayed faster in transgenic mice compared to controls. Microglial activation around motoneurons attenuated faster in parvalbumin overexpressing mice, too, but the decrease of microglial activation was delayed compared to the decline of the chemokine ligand 2 signal. At the time when the microglial reaction peaked, no intracellular calcium increase was detected in the motoneurons of transgenic mice, in contrast to the twofold increase in wild type animals. CONCLUSION: Increased calcium buffering capacity, which augments resistance of motoneurons against calcium-mediated injury, leads to earlier termination of motoneuronal emission of CCL2 followed by a reduction of neighboring microglial activation after axotomy.

Plasticity in Single Axon Glutamatergic Connection to GABAergic Interneurons Regulates Complex Events in the Human Neocortex.

In the human neocortex, single excitatory pyramidal cells can elicit very large glutamatergic EPSPs (VLEs) in inhibitory GABAergic interneurons capable of triggering their firing with short (3-5 ms) delay. Similar strong excitatory connections between two individual neurons have not been found in nonhuman cortices, suggesting that these synapses are specific to human interneurons. The VLEs are crucial for generating neocortical complex events, observed as single pyramidal cell spike-evoked discharge of cell assemblies in the frontal and temporal cortices. However, long-term plasticity of the VLE connections and how the plasticity modulates neocortical complex events has not been studied. Using triple and dual whole-cell recordings from synaptically connected human neocortical layers 2-3 neurons, we show that VLEs in fast-spiking GABAergic interneurons exhibit robust activity-induced long-term depression (LTD). The LTD by single pyramidal cell 40 Hz spike bursts is specific to connections with VLEs, requires group I metabotropic glutamate receptors, and has a presynaptic mechanism. The LTD of VLE connections alters suprathreshold activation of interneurons in the complex events suppressing the discharge of fast-spiking GABAergic cells. The VLEs triggering the complex events may contribute to cognitive processes in the human neocortex, and their long-term plasticity can alter the discharging cortical cell assemblies by learning.

Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically.

We tested the efficacy of treatment with talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in presymptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its efficacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective.

Hypoglossal motor neurons display a reduced calcium increase after axotomy in mice with upregulated parvalbumin.

Motor neurons that exhibit differences in vulnerability to degeneration have been identified in motor neuron disease and in its animal models. The oculomotor and hypoglossal neurons are regarded as the prototypes of the resistant and susceptible cell types, respectively. Because an increase in the level of intracellular calcium has been proposed as a feature amplifying degenerative processes, we earlier studied the calcium increase in these motor neurons after axotomy in Balb/c mice and demonstrated a correlation between the susceptibility to degeneration and the intracellular calcium increase, with an inverse relation with the calcium buffering capacity, characterized by the parvalbumin or calbindin-D(28k) content. Because the differential susceptibility of the cells might also be attributed to their different cellular environments, in the present experiments, with the aim of verifying directly that a higher calcium buffering capacity is indeed responsible for the enhanced resistance, motor neurons were studied in their original milieu in mice with a genetically increased parvalbumin level. The changes in intracellular calcium level of the hypoglossal and oculomotor neurons after axotomy were studied electron microscopically at a 21-day interval after axotomy, during which time no significant calcium increase was detected in the hypoglossal motor neurons, the response being similar to that of the oculomotor neurons. The hypoglossal motor neurons of the parental mice, used as positive controls, exhibited a transient, significant elevation of calcium. These data provide more direct evidence of the protective role of parvalbumin against the degeneration mediated by a calcium increase in the acute injury of motor neurons.