Pharmacological management of gabaergic neurons survival in ischemic conditions: the role of a novel hydroxypyridine compound

Using in vitro models of hypoxia, episodes of short-term hypoxia/reoxygenation, and ischemia-like conditions, we were able to establish different sensitivities of hippocampal neurons to their damaging factors. Using a combination of fluorescence microscopy and immunocytochemistry methods, it was possible to show that GABAergic neurons are the most vulnerable to the damaging effects of hypoxia and ischemia-like conditions, as they have a special metabolism, which is characterized by increased production of reactive oxygen species and nitric oxide. It was shown that long-term hypoxia causes the death of GABAergic neurons due to the induction of a global [Ca2+]i increase, whereas pyramidal neurons are resistant to 30-min hypoxia. Episodes of short-term hypoxia/reoxygenation activate the phenomenon of hypoxic preconditioning in glutamatergic neurons, while this endogenous protective mechanism is absent in GABAergic neurons. Selective activation of PI3K and PKG, combined with NOS inhibition, potentiates the preconditioning effect of hypoxia/reoxygenation in glutamatergic neurons and partially activates this neuroprotective mechanism in GABAergic neurons, as indicated by diminished post-hypoxic NMDA-induced Ca2+ transients. These findings suggest that pharmacological intervention can protect GABAergic neuronal populations. The expression of parvalbumin, calbindin, or calretinin in the cytosol of GABAergic neurons contributes to the suppression of the global [Ca2+]i increase during hypoxia/reoxygenation episodes, which correlates with their survival even in the absence of the hypoxic preconditioning phenomenon. Additionally, all three studied calcium-binding proteins showed potential high efficiency in maintaining the bioenergetics of GABAergic neurons during ischemia-like conditions, limiting ROS production by mitochondria and in the cytosol, and reducing nitric oxide formation. In this case, parvalbumin showed the greatest efficiency.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

Calcium-permeable kainate (CP-KARs) and AMPA (CP-AMPARs) receptors of the brain neurons are active participants of synaptic plasticity and neurotransmitter release trigger. In this paper, CP-KARs and CP-AMPARs were identified in hippocampal neuroglial culture on 14–17 day of cultivation by a characteristic Ca2+ response to a selective agonist of CP-KARs and CP-AMPARs, domoic acid (DA), and to a selective agonist of CP-KARs, ATPA. It was shown that DA at a concentration of 300 nM caused a rapid intracellular Ca2+ concentration increase in two minor subpopulations of neurons. Both subpopulations were found to be GABAergic neurons that were positively stained with antibodies against glutamate decarboxylase 65 and 67 (GAD65/67). The antagonist of CP-AMPARs, NASPM, did not suppress Ca2+ response to DA in the neurons of the first subpopulation. The selective agonist of CP-KARs, ATPA, increased [Ca2+]i to the same extent as DA only in the first subpopulation of GABAergic neurons. An inhibitor of GABA(A) receptors, bicuculline, did not increase the amplitude of Ca2+ response to DA in this subpopulation, indicating the absence of CP-KARs in the postsynaptic membrane, where GABA(A) receptors are located. Thus, these GABAergic neurons can be attributed to neurons containing CP-KARs, which are apparently located in the presynaptic membrane of the GABAergic neurons. The [Ca2+]i increase caused by the DA application in the second subpopulation was completely suppressed by NASPM, an inhibitor of CP-AMPARs. NASPM reduced the Ca2+ oscillations amplitude in the same subset, indicating the involvement of CP-AMPARs in the Ca2+ impulse formation during synchronous calcium activity. For this reason, the neurons of this subpopulation can be attributed to the GABAergic neurons containing CP-AMPARs. Most of the neurons in the hippocampal cell culture (70–85%) were not stained with antibodies against GAD65/67 and responded to the DA by increasing the calcium oscillations frequency with a delay. The amplitude of DA-induced oscillations increased in the presence of NASPM in the subpopulation of inhibitory neurons containing CP-KARs, indicating their innervation by inhibitory neurons containing CP-AMRARs. This increase in the Ca2+ oscillation amplitude in the inhibitory neurons containing CP-KARs correlated with a decrease in the amplitude of synchronous calcium activity in a large subpopulation (42 ± 6% of cells) of glutamatergic neurons, suggesting innervation of the latter by inhibitory neurons containing CP-KARs. Thus, GABAergic neurons containing CP-KARs and CP-AMPARs can work in tandem, controlling the activity of individual subpopulations of neurons.

Corticotropin releasing factor enhances survival of cultured GABAergic cerebellar neurons after exposure to a neurotoxin

Taxifolin protects neurons against ischemic injury in vitro via the activation of antioxidant systems and signal transduction pathways of GABAergic neurons

During normal development of the nervous system, the target fields influence the survival and differentiation of projection neurons, but the factors regulating this interaction remain obscure. In the present study, we have raised the question whether the target region is essential for the postnatal development and maintenance of two different types of central projection neurons, cholinergic and GABAergic septohippocampal cells. In early postnatal rats (P5, P10), the hippocampus was eliminated by unilateral intrahippocampal injections of the excitotoxin N-methyl-D-aspartate. After a long survival time (at P70), we have immunostained serial sections of the septal region with antibodies against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, or the calcium-binding protein parvalbumin (PARV) which is known to be contained in GABAergic septohippocampal neurons. In the medial septum ipsilateral to the lesioned side, about 60% of ChAT-immunoreactive neurons and 62% of PARV-immunoreactive neurons were found in adulthood even after complete elimination of the hippocampus. Some immunoreactive cells appeared heavily shrunken, but electron microscopic analysis revealed ultrastructural characteristics typical for medial septal neurons obtained from controls. Our results indicate that target elimination during development affected both types of projection cells, although only the cholinergic cells are known to be responsive to target-derived factors.

Disturbances in cerebral blood flow cause hypoxic and ischemic processes that lead to damaging and death of neurons. Some populations of GABAergic neurons are characterized by greater sensitivity to oxygen-glucose deprivation. Massive damage and death of the cells (more than 80%) take place in hippocampal cultures during long oxygen-glucose deprivation (40 min). Astrocytes and GABAergic neurons are destroyed first, which in turn leads to the neuroglial network disturbances accompanied by massive death of glutamatergic neurons. In the present work we investigated a protective role of calcium-binding proteins (CaBPs) in the population of GABAergic neurons under hypoxic-like and ischemic-like conditions. The preconditioning was evaluated by suppression of the NMDAR activity after short-term episodes of hypoxia. The posthypoxic hyperexcitability was estimated by the appearance of synchronous spontaneous calcium impulses (s[Ca2+]i) at the reoxigenation stage. The cells damaged during hypoxia and ischemia were detected by the presence of the irreversible increase of [Ca2+]i. The type of neurons and presence of CaBPs (parvalbumin (PV), calbindin (CB), calretinin (CR)) were determined by immunohistochemistry after registration of the [Ca2+]i dynamics. We have shown that any calcium-binding protein in GABAergic neurons can play the role of an endogenous neuroprotector, which prevents calcium overload and subsequent death even without preconditioning. GABAergic neurons containing any CaBP are characterized by lower magnitudes of the calcium responses to the NMDA application. These neurons are not preconditioned by repeated short-term episodes of hypoxia. It was shown that GABAergic neurons containing CR are characterized by the absence of irreversible calcium increases and survive during oxygen-glucose deprivation. However, the presence of PV or CB can lead to the appearance of lag phases with different durations. These two CaBPs reduce the rate of calcium increase and possibly in that way prevent the death of GABAergic neurons under the ischemia-like conditions.

In the rat brain, hippocampal neurons are particularly sensitive to secondary excitotoxic injury induced by ischaemia or hypoglycaemia. To determine some distinctive features of vulnerability among neuronal phenotypes in the hippocampus following a metabolic insult, we used an in vitro model of mild glucose deprivation. Primary cultures from the rat hippocampus (21 days in vitro) were deprived of glucose for 4 h and then were returned to the standard medium for 24 or 48 h. Survival of the GABAergic neuronal population was evaluated both by measuring [3H]GABA uptake and by counting GAD65-immunostained cells. This was compared with the survival of the total neuronal population evaluated by counting the neurofilament-200-immunostained cells. Glucose deprivation for 4 h followed by a recovery period of 48 h induced a decrease of 59% and 40% in the number of GAD65- and neurofilament-200-immunostained cells, respectively. Thus, GABAergic neurons were slightly more vulnerable to glucose deprivation than the other neurons in the hippocampal cell cultures. When the excitotoxic component of cellular death was blocked in the presence of TCP, an NMDA-antagonist, the survival of GABAergic neurons was almost complete after 48 h of recovery. In contrast, measurements of the release of lactate dehydrogenase in the medium indicated that TCP largely protected hippocampal cells after 24 h but was ineffective after 48 h. This observation was confirmed by immunostaining data which showed that after 48 h TCP did not significantly increase the survival of neurofilament-200-immunostained cells. These results indicate that after glucose deprivation and a recovery period of 48 h, GABAergic neurons in hippocampal cell cultures are not more resistant than other neurons but they are more sensitive to TCP protection.

Marco Capogna, a pioneering neuroscientist and true European

The physiological responses of dendrites to dopaminergic inputs are poorly understood and controversial. We applied dopamine on one dendritic branch while simultaneously monitoring action potentials (APs) from multiple dendrites using either calcium-sensitive dye, voltage-sensitive dye or both. Dopaminergic suppression of dendritic calcium transients was rapid (<0.5 s) and restricted to the site of dopamine application. Voltage waveforms of backpropagating APs were minimally altered in the same dendrites where dopamine was confirmed to cause large suppression of calcium signals, as determined by dual voltage and calcium imaging. The dopamine effects on dendritic calcium transients were fully mimicked by D1 agonists, partially reduced by D1 antagonist and completely insensitive to protein kinase blockade; consistent with a membrane delimited mechanism. This dopamine effect was unaltered in the presence of L-, R- and T-type calcium channel blockers. The somatic excitability (i.e. AP firing) was not affected by strong dopaminergic stimulation of dendrites. Dopamine and GABA were then sequentially applied on the same dendrite. In contrast to dopamine, the pulses of GABA prohibited AP backpropagation distally from the application site, even in neurons with natural Cl− concentration (patch pipette removed). Thus, the neocortex employs at least two distinct mechanisms (dopamine and GABA) for rapid modulation of dendritic calcium influx. The spatio-temporal pattern of dendritic calcium suppression described in this paper is expected to occur during phasic dopaminergic signalling, when midbrain dopaminergic neurons generate a transient (0.5 s) burst of APs in response to a salient event.

Front cover Calcium-permeable AMPA receptors (CP-AMPARs) are involved in synaptogenesis and formation of neuronal circuitry. However, these receptors provide additional Ca2+ inflow into neurons, contributing dysregulation of Ca2+ homeostasis and neuronal dysfunction in excitotoxicity associated with numerous CNS disorders. Considering these aspects of Ca2+ activity, we have proposed the method of vital identification of neurons containing CP-AMPARs. Using this method we demonstrate that approximately 80% CP-AMPARs-containing neurons in rat hippocampal cell cultures are GABAergic. As with most GABAergic neurons, GABAergic neurons containing CP-AMPARs demonstrate weaker GABA(A)R-mediated innervation compared to glutamatergic neurons. This peculiarity together with the presence of CP-AMPARs may contribute to vulnerability of these neurons to excitotoxicity. Image content Microphotographs of neurons in rat hippocampal neuron-glial culture stained with antibodies against GABA (red) and a marker of a GABAergic synapse, vesicular GABA transporter (green puncta). The panel shows the distribution of GABAergic synapses on two glutamatergic neurons (left image) and GABAergic neuron with (right image) and without CP-AMPARs (middle image). Read the full article ‘A novel approach for vital visualization and studying of neurons containing Ca2+-permeable AMPA receptors’ by S. G. Gaidin, S. A. Maiorov, D. P. Laryushkin, V. P. Zinchenko and A. M. Kosenkov, (J. Neurochem. 2023, vol.164 (5), pp. 583–587) on doi:10.1111/jnc.15729

The effect of the depolarizing agents, an elevated potassium concentration (25 mM) or kainic acid (50 microM) on neuronal survival and differentiation was investigated in cultures of dissociated neurons from cerebella of 7-day-old mice. When maintained in the presence of an antimitotic agent such cultures consist primarily of glutamatergic and GABAergic neurons. Cell survival was monitored by measurement of DNA, and differentiation by determining uptake and depolarization coupled release of glutamate (D-aspartate as label) and GABA. The depolarizing agents were added separately or together either from the start of the culture period (7-8 days) or at day 5 in culture. The main findings are that K+ depolarization is important for differentiation of glutamatergic neurons but not for GABAergic neurons. This depolarizing signal is important during the early phase of development in culture. For glutamatergic neurons, kainate may replace K+ as a depolarizing signal whereas in case of the GABAergic neurons, kainate was toxic particularly during the late phase of development. It was further observed that the glutamatergic neurons when maintained in a medium with 5 mM K+ during the first 5 days in culture became sensitive to kainate toxicity when this amino acid was added at day 5. This was not the case when the medium contained 25 mM K+ from the start of the culture period.

Late appearance of parvalbumin-immunoreactivity in the development of GABAergic neurons in the rat hippocampus

Stress in the endoplasmic reticulum caused by tunicamycin, dithiothreitol, and azole-class antifungal drugs can induce nonapoptotic cell death in yeasts that can be blocked by the action of calcineurin (Cn), a Ca(2+)-dependent serine/threonine protein phosphatase. To identify additional factors that regulate nonapoptotic cell death in yeast, a collection of gene knock-out mutants was screened for mutants exhibiting altered survival rates. The screen revealed an endocytic protein (Ede1) that can function upstream of Ca(2+)/calmodulin-dependent protein kinase 2 (Cmk2) to suppress cell death in parallel to Cn. The screen also revealed the vacuolar H(+)-ATPase (V-ATPase), which acidifies the lysosome-like vacuole. The V-ATPase performed its death-promoting functions very soon after imposition of the stress and was not required for later stages of the cell death program. Cn did not inhibit V-ATPase activities but did block vacuole membrane permeabilization (VMP), which occurred at late stages of the cell death program. All of the other nondying mutants identified in the screens blocked steps before VMP. These findings suggest that VMP is the lethal event in dying yeast cells and that fungi may employ a mechanism of cell death similar to the necrosis-like cell death of degenerating neurons.

In the present study, we have investigated the developmental expression of the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium-binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons. A large number of GAD67 mRNA-expressing neurons were already observed in the septal complex on embryonic day (E) 17, the earliest time point studied. During later developmental stages, there was mainly an increase in the intensity of labeling. Neurons expressing ChAT mRNA were first recognized at E 20, and their number slowly increased during postnatal development of the septal region. The adult pattern of ChAT mRNA-expressing neurons was observed around postnatal day (P) 16. By using a monoclonal ChAT antibody, the first immunoreactive cells were not seen before P 8. Similarly, the first weakly parvalbumin-immunoreactive neurons were seen in the septal complex by the end of the 1st postnatal week. These results indicate that in situ hybridization histochemistry may be an adequate method to monitor the different development of transmitter biosynthesis in cholinergic and GABAergic septal neurons. Moreover, the late onset of ChAT mRNA expression would be compatible with a role of target-derived factors for the differentiation of the cholinergic phenotype.