Karl W. Kafitz

Neurotrophin-evoked rapid excitation through TrkB receptors

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Developmental profile and properties of sulforhodamine 101—Labeled glial cells in acute brain slices of rat hippocampus

The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31-7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24-27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na+ currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.

Developmental profile and mechanisms of GABA-induced calcium signaling in hippocampal astrocytes

GABA (γ‐aminobutyric acid) is a transmitter with dual action. Whereas it excites neurons during the first week of postnatal development, it represents the major inhibitory transmitter in the mature brain. GABA also activates astrocytes by binding to ionotropic (GABAA) and metabotropic (GABAB) receptors. This results in glial calcium transients which can induce the release of gliotransmitters, rendering GABA an important mediator of neuron‐glia interaction. Using whole‐cell patch‐clamp and ratiometric calcium imaging in hippocampal slices from rats at postnatal days 3–34, we have analyzed the developmental profile as well as the cellular mechanisms of calcium signals induced by GABAA and GABAB receptor activation in astrocytes. We found that GABA‐evoked glial calcium transients are mediated by both GABAA and GABAB receptors. Throughout development, GABAA‐receptor activation resulted in immediate calcium transients in the vast majority of astrocytes, most likely by influx of calcium through voltage‐gated calcium channels. GABAB receptor activation, in contrast, resulted in delayed calcium transients, which were blocked following depletion of intracellular calcium stores and during persistent activation of heterotrimeric G‐proteins. GABAB receptor‐mediated calcium signals exhibited a clear developmental profile with less than 10% of astrocytes responding at P3 or P32–34, and about 60% of cells between P11 and P15. Our data thus indicate that GABAB receptor‐mediated calcium transients are due to calcium release from intracellular stores following G‐protein activation. Moreover, GABAB receptor‐mediated calcium signaling in astrocytes preferentially occurs at a period during postnatal development when hippocampal networks are established.

Ammonium-evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH4+/NH3). Here, we examined the effects of NH4+/NH3 on steady‐state intracellular pH (pHi) and sodium concentration ([Na+]i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH4+/NH3 on glutamate transporter activity by measuring D‐aspartate‐induced pHi and [Na+]i transients. In 20–34 days in vitro (DIV) astrocytes, NH4+/NH3 decreased steady‐state pHi by 0.19 pH units and increased [Na+]i by 21 mM. D‐Aspartate‐induced pHi and [Na+]i transients were reduced by 80–90% in the presence of NH4+/NH3, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pHi and [Na+]i were minimally affected by NH4+/NH3, and D‐aspartate‐induced pHi and [Na+]i transients were reduced by only 30–40%. Next we determined the contribution of Na+, K+, Cl−‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH4+/NH3‐evoked changes in steady‐state pHi and [Na+]i and attenuated the reduction of D‐aspartate‐induced pHi and [Na+]i transients by NH4+/NH3 to 30% in 20–34 DIV astrocytes. Our results suggest that NH4+/NH3 decreases steady‐state pHi and increases steady‐state [Na+]i in astrocytes by an age‐dependent activation of NKCC. These NH4+/NH3‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission.

Glial cells express specific high‐affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole‐cell patch‐clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium‐dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.

Astrocyte Calcium Signals at Schaffer Collateral to CA1 Pyramidal Cell Synapses Correlate With the Number of Activated Synapses But Not With Synaptic Strength

Glial cells respond to neuronal activity by transient increases in their intracellular calcium concentration. At hippocampal Schaffer collateral to CA1 pyramidal cell synapses, such activity‐induced astrocyte calcium transients modulate neuronal excitability, synaptic activity, and LTP induction threshold by calcium‐dependent release of gliotransmitters. Despite a significant role of astrocyte calcium signaling in plasticity of these synapses, little is known about activity‐dependent changes of astrocyte calcium signaling itself. In this study, we analyzed calcium transients in identified astrocytes and NG2‐cells located in the stratum radiatum in response to different intensities and patterns of Schaffer collateral stimulation. To this end, we employed multiphoton calcium imaging with the low‐affinity indicator dye Fluo‐5F in glial cells, combined with extracellular field potential recordings to monitor postsynaptic responses to the afferent stimulation. Our results confirm that somata and processes of astrocytes, but not of NG2‐cells, exhibit intrinsic calcium signaling independent of evoked neuronal activity. Moderate stimulation of Schaffer collaterals (three pulses at 50 Hz) induced calcium transients in astrocytes and NG2‐cells. Astrocyte calcium transients upon this three‐pulse stimulation could be evoked repetitively, increased in amplitude with increasing stimulation intensity and were dependent on activation of metabotropic glutamate receptors. Activity‐induced transients in NG2‐cells, in contrast, showed a rapid run‐down upon repeated three‐pulse stimulation. Theta burst stimulation and stimulation for 5 min at 1 Hz induced synaptic potentiation and depression, respectively, as revealed by a lasting increase or decrease in population spike amplitudes upon three‐pulse stimulation. Synaptic plasticity was, however, not accompanied by corresponding alterations in the amplitude of astrocyte calcium signals. Taken together, our results suggest that the amplitude of astrocyte calcium signals reflects the number of activated synapses but does not correlate with the degree of synaptic potentiation or depression at Schaffer collateral to CA1 pyramidal cell synapses.

Laminar and subcellular heterogeneity of GLAST and GLT-1 immunoreactivity in the developing postnatal mouse hippocampus

Astrocytes express two sodium‐coupled transporters, glutamate–aspartate transporter (GLAST) and glutamate transporter‐1 (GLT‐1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT‐1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high‐resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP‐positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT‐1 was completed only at postnatal days (P) P20–25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3–5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT‐1 exhibited a laminar expression pattern from P10–15 on, with the highest immunoreactivity in the stratum lacunosum‐moleculare. At the cellular level, GLT‐1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT‐1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT‐1 expression in the developing hippocampus. The clustering of GLT‐1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood–brain barrier during formation of the hippocampal network. J. Comp. Neurol. 522:204–224, 2014.

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood–brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate‐induced endfeet sodium transients were diminished by TFB‐TBOA, suggesting that they were generated by sodium‐dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT‐1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double‐deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long‐lived and inter‐cellularly acting indicator of synaptic activity at the blood–brain barrier, which likely constitutes an important component of neuro‐metabolic coupling in the brain. GLIA 2017;65:293–308

Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels.

Corticotropin-releasing hormone (CRH) plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS) and field excitatory postsynaptic potentials (fEPSP) were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean ± Standard error of the mean; 231.8 ± 31.2% of control; n = 10) while neither affecting fEPSPs (104.3 ± 4.2%; n = 10) nor long-term potentiation (LTP). However, when Schaffer-collaterals were excited via action potentials (APs) generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n = 8) and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1) expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

Lesion-Induced Alterations in Astrocyte Glutamate Transporter Expression and Function in the Hippocampus

Astrocytes express the sodium-dependent glutamate transporters GLAST and GLT-1, which are critical to maintain low extracellular glutamate concentrations. Here, we analyzed changes in their expression and function following a mechanical lesion in the CA1 area of organotypic hippocampal slices. 6-7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased GFAP and S100β immunoreactivity, enlarged somata, and reduced capability for uptake of SR101. Astrocytes in the scars periphery were swollen as well, but showed only moderate upregulation of GFAP and S100β and efficiently took up SR101. In the scar, clusters of GLT-1 and GLAST immunoreactivity colocalized with GFAP-positive fibers. Apart from these, GLT-1 immunoreactivity declined with increasing distance from the scar, whereas GLAST expression appeared largely uniform. Sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. Our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. Strongly reactive astrocytes in the scar, however, display clusters of GLAST and GLT-1 immunoreactivity together with reduced glutamate transport activity. This reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site.