Distinct brain-wide presynaptic networks underlie the functional identity of individual cortical neurons.

Neuronal connections provide the scaffolding for neuronal function. Revealing the connectivity of functionally identified individual neurons is necessary to understand how activity patterns emerge and support behavior. Yet, the brain-wide presynaptic wiring rules that lay the foundation for the functional selectivity of individual neurons remain largely unexplored. Cortical neurons, even in primary sensory cortex, are heterogeneous in their selectivity, not only to sensory stimuli but also to multiple aspects of behavior. Here, to investigate presynaptic connectivity rules underlying the selectivity of pyramidal neurons to behavioral state 1-12 in primary somatosensory cortex (S1), we used two-photon calcium imaging, neuropharmacology, single-cell based monosynaptic input tracing, and optogenetics. We show that behavioral state-dependent neuronal activity patterns are stable over time. These are not determined by neuromodulatory inputs but are instead driven by glutamatergic inputs. Analysis of brain-wide presynaptic networks of individual neurons with distinct behavioral state-dependent activity profiles revealed characteristic patterns of anatomical input. While both behavioral state-related and unrelated neurons had a similar pattern of local inputs within S1, their long-range glutamatergic inputs differed. Individual cortical neurons, irrespective of their functional properties, received converging inputs from the main S1-projecting areas. Yet, neurons that tracked behavioral state received a smaller proportion of motor cortical inputs and a larger proportion of thalamic inputs. Optogenetic suppression of thalamic inputs reduced behavioral state-dependent activity in S1, but this activity was not externally driven. Our results revealed distinct long-range glutamatergic inputs as a substrate for preconfigured network dynamics associated with behavioral state.

Posteromedial thalamic nucleus activity significantly contributes to perceptual discrimination.

Higher-order sensory thalamic nuclei are densely connected with multiple cortical and subcortical areas, yet the role of these nuclei remains elusive. The posteromedial thalamic nucleus (POm), the higher-order thalamic nucleus in the rodent somatosensory system, is an anatomical hub broadly connected with multiple sensory and motor brain areas yet weakly responds to passive sensory stimulation and whisker movements. To understand the role of POm in sensory perception, we developed a self-initiated, two-alternative forced-choice task in freely moving mice during active sensing. Using optogenetic and chemogenetic manipulation, we show that POm plays a significant role in sensory perception and the projection from the primary somatosensory cortex to POm is critical for the contribution of POm in sensory perception during active sensing.

Sensory feedback synchronizes motor and sensory neuronal networks in the neonatal rat spinal cord.

Early stages of sensorimotor system development in mammals are characterized by the occurrence of spontaneous movements. Whether and how these movements support correlated activity in developing sensorimotor spinal cord circuits remains unknown. Here we show highly correlated activity in sensory and motor zones in the spinal cord of neonatal rats in vivo. Both during twitches and complex movements, movement-generating bursts in motor zones are followed by bursts in sensory zones. Deafferentation does not affect activity in motor zones and movements, but profoundly suppresses activity bursts in sensory laminae and results in sensorimotor uncoupling, implying a primary role of sensory feedback in sensorimotor synchronization. This is further supported by largely dissociated activity in sensory and motor zones observed in the isolated spinal cord in vitro. Thus, sensory feedback resulting from spontaneous movements is instrumental for coordination of activity in developing sensorimotor spinal cord circuits.

Endogenous IFN-ß signaling exerts anti-inflammatory actions in experimentally induced focal cerebral ischemia.

BACKGROUND: Interferon (IFN)-ß exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-ß, by limiting inflammation, decreases neuronal death and promotes functional recovery after stroke. However, the core actions of endogenous IFN-ß signaling in stroke are unclear. METHODS: To address this question, we used two clinically relevant models of focal cerebral ischemia, transient and permanent middle cerebral artery occlusion, and two genetically modified mouse lines, lacking either IFN-ß or its receptor, the IFN-α/ß receptor. Subsets of inflammatory and immune cells isolated from the brain, blood, and spleen were studied using flow cytometry. Sensorimotor deficits were assessed by a modified composite neuroscore, the rotating pole and grip strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. RESULTS: Here, we report alterations in local and systemic inflammation in IFN-ß knockout (IFN-ßKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-ßKO mice showed a higher number of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-ßKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation of circulating cytokines and splenic immune cell populations in wild-type and IFN-ßKO mice further supports an important immunoregulatory function of IFN-ß in stroke. Moreover, we observed a significant weight loss 2-3 days and a reduction in grip strength 2 days after stroke in the IFN-ßKO group, while endogenous IFN-ß signaling did not affect the infarct volume. CONCLUSIONS: We conclude that endogenous IFN-ß signaling attenuates local inflammation, regulates peripheral immune cells, and, thereby, may contribute positively to stroke outcome.

Isoflurane suppresses early cortical activity.

OBJECTIVE: Isoflurane and other volatile anesthetics are widely used in children to induce deep and reversible coma, but they may also exert neurotoxic actions. The effects of volatile anesthetics on the immature brain activity remain elusive, however. METHODS: The effects of isoflurane on spontaneous and sensory-evoked activity were explored using intracortical extracellular field potential and multiple unit recordings in the rat barrel cortex from birth to adulthood. RESULTS: During the first postnatal week, isoflurane suppressed cortical activity in a concentration-dependent manner. At surgical anesthesia levels (1.5-2%), isoflurane completely suppressed the electroencephalogram and silenced cortical neurons. Although sensory potentials evoked by the principal whisker deflection persisted, sensory-evoked early gamma and spindle-burst oscillations were completely suppressed by isoflurane. Isoflurane-induced burst-suppression pattern emerged during the second postnatal week and matured through the first postnatal month. Bursts in adolescent and adult rats were characterized by activation of entire cortical columns with a leading firing of infragranular neurons, and were triggered by principal and adjacent whiskers stimulation, and by auditory and visual stimuli, indicating an involvement of horizontal connections in their generation and horizontal spread. INTERPRETATION: The effects of isoflurane on cortical activity shift from total suppression of activity to burst-suppression pattern at the end of the first postnatal week. Developmental emergence of bursts likely involves a development of the intracortical short-and long-range connections. We hypothesize that complete suppression of cortical activity under isoflurane anesthesia during the first postnatal week may explain neuronal apoptosis stimulated by volatile anesthetics in the neonatal rats.

GABA(A) receptor dephosphorylation followed by internalization is coupled to neuronal death in in vitro ischemia.

Cerebral ischemia is characterized by an early disruption of GABAergic neurotransmission contributing to an imbalance of the excitatory/inhibitory equilibrium and neuronal death, but the molecular mechanisms involved are not fully understood. Here we report a downregulation of GABA(A) receptor (GABA(A)R) expression, affecting both mRNA and protein levels of GABA(A)R subunits, in hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Similar alterations in the abundance of GABA(A)R subunits were observed in in vivo brain ischemia. OGD reduced the interaction of surface GABA(A)R with the scaffold protein gephyrin, followed by clathrin-dependent receptor internalization. Internalization of GABA(A)R was dependent on glutamate receptor activation and mediated by dephosphorylation of the ß3 subunit at serine 408/409. Expression of phospho-mimetic mutant GABA(A)R ß3 subunits prevented receptor internalization and protected hippocampal neurons from ischemic cell death. The results show a key role for ß3 GABA(A)R subunit dephosphorylation in the downregulation of GABAergic synaptic transmission in brain ischemia, contributing to neuronal death. GABA(A)R phosphorylation might be a therapeutic target to preserve synaptic inhibition in brain ischemia.

Effects of the sigma-1 receptor agonist 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)-piperazine dihydro-chloride on inflammation after stroke.

Activation of the sigma-1 receptor (Sig-1R) improves functional recovery in models of experimental stroke and is known to modulate microglia function. The present study was conducted to investigate if Sig-1R activation after experimental stroke affects mediators of the inflammatory response in the ischemic hemisphere. Male Wistar rats were subjected to transient occlusion of the middle cerebral artery (MCAO) and injected with the specific Sig-1R agonist 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (SA4503) or saline for 5 days starting on day 2 after MCAO. Treatment did not affect the increased levels of the pro-inflammatory cytokines interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13) in the infarct core and peri-infarct area after MCAO. In addition, treatment with SA4503 did not affect elevated levels of nitrite, TNF-α and IL-1ß observed in primary cultures of microglia exposed to combined Hypoxia/Aglycemia, while the unspecific sigma receptor ligand 1,3-di-o-tolylguanidine (DTG) significantly decreased the production of nitrite and levels of TNF-α. Analysis of the ischemic hemisphere also revealed increased levels of ionized calcium binding adaptor molecule 1 (Iba1) levels in the infarct core of SA4503 treated animals. However, no difference in Iba1 immunoreactivity was detected in the infarct core. Also, levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and OX-42 were not increased in the infarct core in rats treated with SA4503. Together, our results suggest that sigma-1 receptor activation affects Iba1 expression in microglia/macrophages of the ischemic hemisphere after experimental stroke but does not affect post-stroke inflammatory mediators.

Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival through activation of TrkB receptors. The trkB gene encodes a full-length receptor tyrosine kinase (TrkB.FL) and its truncated (T1/T2) isoforms. We investigated the changes in TrkB protein levels and signaling activity under excitotoxic conditions, which are characteristic of brain ischemia, traumatic brain injury, and neurodegenerative disorders. Excitotoxic stimulation of cultured rat hippocampal or striatal neurons downregulated TrkB.FL and upregulated a truncated form of the receptor (TrkB.T). Downregulation of TrkB.FL was mediated by calpains, whereas the increase in TrkB.T protein levels required transcription and translation activities. Downregulation of TrkB.FL receptors in hippocampal neurons correlated with a decrease in BDNF-induced activation of the Ras/ERK and PLCγ pathways. However, calpain inhibition, which prevents TrkB.FL degradation, did not preclude the decrease in signaling activity of these receptors. On the other hand, incubation with anisomycin, to prevent the upregulation of TrkB.T, protected to a large extent the TrkB.FL signaling activity, suggesting that truncated receptors may act as dominant-negatives. The upregulation of TrkB.T under excitotoxic conditions was correlated with an increase in BDNF-induced inhibition of RhoA, a mediator of excitotoxic neuronal death. BDNF fully protected hippocampal neurons transduced with TrkB.T when present during excitotoxic stimulation with glutamate, in contrast with the partial protection observed in cells overexpressing TrkB.FL or expressing GFP. These results indicate that BDNF protects hippocampal neurons by two distinct mechanisms: through the neurotrophic effects of TrkB.FL receptors and by activation of TrkB.T receptors coupled to inhibition of the excitotoxic signaling.

Cleavage of the vesicular glutamate transporters under excitotoxic conditions.

Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.

Lack of macrophage migration inhibitory factor in mice does not affect hallmarks of the inflammatory/immune response during the first week after stroke.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been proposed to play a detrimental role in stroke. We recently showed that MIF promotes neuronal death and aggravates neurological deficits during the first week after experimental stroke, in mice. Since MIF regulates tissue inflammation, we studied the putative role of MIF in post-stroke inflammation. METHODS: We subjected C57BL/6 mice, Mif-/- (MIF-KO) or Mif+/+ (WT), to a transient occlusion of the right middle cerebral artery (tMCAo) or sham-surgery. We studied MIF expression, GFAP expression and the number of CD74-positive cells in the ischemic brain hemisphere 7 days after tMCAo using primarily immunohistochemistry. We determined IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, KC/CXCL-1 and TNF-α protein levels in the brain (48 h after surgery) and serum (48 h and 7 days after surgery) by a multiplex immunoassay. RESULTS: We observed that MIF accumulates in neurons and astrocytes of the peri-infarct region, as well as in microglia/macrophages of the infarct core up to 7 days after stroke. Among the inflammatory mediators analyzed, we found a significant increase in cerebral IL-12 and KC levels after tMCAo, in comparison to sham-surgery. Importantly, the deletion of Mif did not significantly affect the levels of the cytokines evaluated, in the brain or serum. Moreover, the spleen weight 48 h and 7 days subsequent to tMCAo was similar in WT and MIF-KO mice. Finally, the extent of GFAP immunoreactivity and the number of MIF receptor (CD74)-positive cells within the ischemic brain hemisphere did not differ significantly between WT and MIF-KO mice subjected to tMCAo. CONCLUSIONS: We conclude that MIF does not affect major components of the inflammatory/immune response during the first week after experimental stroke. Based on present and previous evidence, we propose that the deleterious MIF-mediated effects in stroke depend primarily on an intraneuronal and/or interneuronal action.

Cleavage of the vesicular GABA transporter under excitotoxic conditions is followed by accumulation of the truncated transporter in nonsynaptic sites.

GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.

Enriched environment downregulates macrophage migration inhibitory factor and increases parvalbumin in the brain following experimental stroke.

Housing rodents in an enriched environment (EE) following experimental stroke enhances neurological recovery. Understanding the underlying neural cues may provide the basis for improving stroke rehabilitation. We studied the contribution of brain macrophage migration inhibitory factor (MIF) to functional recovery after permanent middle cerebral artery occlusion (pMCAo) in rats. In the cerebral cortex, MIF is predominantly found in neurons, particularly in parvalbumin interneurons. Following pMCAo, MIF increases around the infarct core, where it is located to neurons and astrocytes. Housing rats in an EE after pMCAo resulted in a decrease of MIF protein levels in peri-infarct areas, which was accompanied by an increase in parvalbumin immunoreactive interneurons. Our data suggest that MIF is part of a signaling network involved in brain plasticity, and elevated neuronal and/or astrocytic MIF levels repress the recovery of sensory-motor function after stroke. Downregulating MIF could constitute a new therapeutic approach to promote recovery after stroke.

Macrophage migration inhibitory factor promotes cell death and aggravates neurologic deficits after experimental stroke.

Multiple mechanisms contribute to tissue demise and functional recovery after stroke. We studied the involvement of macrophage migration inhibitory factor (MIF) in cell death and development of neurologic deficits after experimental stroke. Macrophage migration inhibitory factor is upregulated in the brain after cerebral ischemia, and disruption of the Mif gene in mice leads to a smaller infarct volume and better sensory-motor function after transient middle cerebral artery occlusion (tMCAo). In mice subjected to tMCAo, we found that MIF accumulates in neurons of the peri-infarct region, particularly in cortical parvalbumin-positive interneurons. Likewise, in cultured cortical neurons exposed to oxygen and glucose deprivation, MIF levels increase, and inhibition of MIF by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) protects against cell death. Deletion of MIF in Mif(-/-) mice does not affect interleukin-1ß protein levels in the brain and serum after tMCAo. Furthermore, disruption of the Mif gene in mice does not affect CD68, but it is associated with higher galectin-3 immunoreactivity in the brain after tMCAo, suggesting that MIF affects the molecular/cellular composition of the macrophages/microglia response after experimental stroke. We conclude that MIF promotes neuronal death and aggravates neurologic deficits after experimental stroke, which implicates MIF in the pathogenesis of neuronal injury after stroke.

Effects of chronic Clozapine administration on apolipoprotein D levels and on functional recovery following experimental stroke.

Elevated brain levels of apolipoprotein D (ApoD) correlate with improved neurological recovery after experimental stroke. Hence, a pharmacological induction of ApoD in the postischemic brain could be beneficial for recovery after stroke. Here we investigated the effect of Clozapine, a compound that increases the expression of ApoD, in two rat models of experimental stroke. Rats were subjected to permanent occlusion of the middle cerebral artery (pMCAO) and treated with Clozapine (i.p. 10 mg/kg body weight) or saline for 8 or 28 days starting on the second day after MCAO. ApoD levels increased by 35% in the peri-infarct area after 10 and 30 days after pMCAO, mainly in neuron-specific nuclear protein (NeuN) positive neurons and glial fibrillary acidic protein (GFAP) positive astrocytes. Clozapine did not affect the neurological deficit assessed by the rotating pole test and a grip strength test at 7 days, 14 days, 21 days, and 28 days after pMCAO. Functional outcome and the infarct size were similar in rats subjected to transient MCAO and injected with Clozapine (i.p. 10 mg/kg body weight) or saline for 26 days starting on the second day after tMCAO. We conclude that Clozapine affects cellular processes involved in peri-infarct tissue reorganization, but does not affect functional recovery after MCAO.