Mounting evidence indicates that microglial activation contributes to neuronal damage in neurodegenerative diseases. Recent studies show that in response to certain environmental toxins and endogenous proteins, microglia can enter an overactivated state and release reactive oxygen species (ROS) that cause neurotoxicity. Pattern recognition receptors expressed on the microglial surface seem to be one of the primary, common pathways by which diverse toxin signals are transduced into ROS production. Overactivated microglia can be detected using imaging techniques and therefore this knowledge offers an opportunity not only for early diagnosis but, importantly, for the development of targeted anti-inflammatory therapies that might slow or halt the progression of neurodegenerative disease.

Microglia-mediated neurotoxicity: uncovering the molecular mechanisms

Microglial cells are the resident macrophages in the central nervous system. These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system, disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed "resting microglia." Recent studies indicate that even in the normal brain, microglia have highly motile processes by which they scan their territorial domains. By a large number of signaling pathways they can communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells express receptors classically described for brain-specific communication such as neurotransmitter receptors and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the "activated microglial cell." This cell form has the capacity to release a large number of substances that can act detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury, proliferate, and phagocytose cells and cellular compartments.

Physiology of Microglia

This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

In many neurologic disorders, injury to neurons may be caused at least in part by overstimulation of receptors for excitatory amino acids, including glutamate and aspartate. These neurologic conditions range from acute insults such as stroke, hypoglycemia, trauma, and epilepsy (Table 1) to chronic neurodegenerative states such as Huntington's disease, the acquired immunodeficiency syndrome (AIDS) dementia complex, amyotrophic lateral sclerosis, and perhaps Alzheimer's disease (Table 2)1–3. Glutamate is the principal excitatory neurotransmitter in the brain, and its interactions with specific membrane receptors are responsible for many neurologic functions, including cognition, memory, movement, and sensation4. In addition, excitatory . . .

Excitatory amino acids as a final common pathway for neurologic disorders.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

https://pharmrev.aspetjournals.org/content/pharmrev/54/2/161.full.pdf

International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology Nitrendipine inhibited activity of L-type channels but not N-type channels Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2 Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine

https://science.sciencemag.org/content/sci/239/4835/57.full.pdf

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Activation of cannabinoid receptors inhibits voltage-gated Ca2+ channels and activates K+ channels, reminiscent of other G-protein-coupled signaling pathways that produce presynaptic inhibition. We tested cannabinoid receptor agonists for effects on excitatory neurotransmission between cultured rat hippocampal neurons. Reducing the extracellular Mg2+ concentration to 0.1 mM elicited repetitive, transient increases in intracellular Ca2+ concentration ([Ca2+]i spikes) that resulted from bursts of action potentials, as measured by combined whole-cell current clamp and indo-1-based microfluorimetry. Pharmacological characterization indicated that the [Ca2+]i spikes required glutamatergic synaptic transmission. Cannabinoid receptor ligands inhibited stereoselectively the frequency of [Ca2+]i spiking in the rank order of potency: CP 54,939 > CP 55,940 > Win 55,212-2 > anandamide, with EC50 values of 0.36, 1.2, 2.7, and 71 nM, respectively. CP 55,940 was potent, but not efficacious, and reversed the inhibition produced by Win 55,212-2, indicating that it is a partial agonist. Inhibition of [Ca2+]i spiking by Win 55,212-2 was prevented by treatment of cultures with active, but not heat-treated, pertussis toxin. Win 55,212-2 (100 nM) inhibited stereoselectively CNQX-sensitive excitatory postsynaptic currents (EPSCs) elicited by presynaptic stimulation with an extracellular electrode, but did not affect the presynaptic action potential or currents elicited by direct application of kainate. Consistent with a presynaptic site of action, Win 55,212-2 increased both the number of response failures and the coefficient of variation of the evoked EPSCs. In contrast, cannabimimetics did not affect bicuculline-sensitive inhibitory postsynaptic currents. Thus, activation of cannabinoid receptors inhibits the presynaptic release of glutamate via an inhibitory G-protein.

https://www.jneurosci.org/content/jneuro/16/14/4322.full.pdf

Cannabinoid Receptor Agonists Inhibit Glutamatergic Synaptic Transmission in Rat Hippocampal Cultures

We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons Intracellular free calcium concentration ([Ca2+]i) and intracellular free hydrogen ion concentration ([H+]i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry Field potential stimulation evoked action potential-mediated increases in [Ca2+] Brief trains of action potentials elicited [Ca2+]i transients that recovered to basal levels by a single exponential process Trains of > 25 action potentials elicited larger increases in [Ca2+]i, recovery from which consisted of three distinct phases During a rapid initial phase [Ca2+]i decreased to a plateau level (450-550 nM) The plateau was followed by a slow return to basal [Ca2+]i [Ca2+]i transients elicited by 40-50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal [Ca2+]i consisted of a single slow phase Depolarization with 50 mM K+ produced a multiphasic [Ca2+]i transient and increased [H+]i from 74 +/- 3 to 107 +/- 8 nM The rise in [H+]i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal [Ca2+]i was unaffected by removal of extracellular Na+ We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in [H+]i Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)-independent process

https://www.jneurosci.org/content/jneuro/14/1/348.full.pdf

Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons

Glutamate-induced changes in intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single rat hippocampal neurons grown in primary culture by employing the Ca2+ indicator indo-1 and a dual- emission microfluorimeter. The [Ca2+]i was monitored in neurons exposed to 100 microM glutamate for 5 min and for an ensuing 3 hr period. Ninety-two percent (n = 64) of these neurons buffered the glutamate- induced Ca2+ load back to basal levels after removal of the agonist; thus, the majority of cells had not lost the ability to regulate [Ca2+]i at this time. However, following a variable delay, in 44% (n = 26) of the neurons that buffered glutamate-induced Ca2+ loads to basal levels, [Ca2+]i rose again to a sustained plateau and failed to recover. The changes in [Ca2+]i that occur during glutamate-induced delayed neuronal death can be divided into three phases: (1) a triggering phase during which the neuron is exposed to glutamate and the [Ca2+]i increases to micromolar levels, followed by (2) a latent phase during which the [Ca2+]i recovers to a basal level, and (3) a final phase that begins with a gradual rise in the [Ca2+]i that reaches a sustained plateau from which the neuron does not recover. This delayed Ca2+ overload phase correlated significantly with cell death. The same sequence of events was also observed in recordings from neuronal processes. The delayed Ca2+ increase and subsequent death were dependent upon the presence of extracellular Ca2+ during glutamate exposure. Calcium influx during the triggering phase resulted from the activation of both NMDA and non-NMDA receptors as indicated by studies using receptor antagonists and ion substitution. Treatment with TTX (1 microM) or removal of extracellular Ca2+ for a 30 min window following agonist exposure failed to prevent the delayed Ca2+ overload. The delayed [Ca2+]i increase could be reversed by removing extracellular Ca2+, indicating that it resulted from Ca2+ influx. The three phases defined by changes in the [Ca2+]i during glutamate-induced neuronal toxicity suggest three distinct targets to which neuroprotective agents may be directed.

https://www.jneurosci.org/content/jneuro/12/5/1882.full.pdf

Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons

1. Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an [Ca2+]i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered [Ca2+]i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and [Ca2+]i relationship revealed that the peak of the [Ca2+]i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2(+)-induced Ca2+ release from intracellular stores made a significant contribution to the [Ca2+]i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, [Ca2+]i buffering was more effective for larger Ca2+ loads. The relationship between the integrated ICa and the peak of the [Ca2+]i transient reached an asymptote at large Ca2+ loads indicating that Ca2(+)-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the [Ca2+]i transient, while inhibition of smaller currents produced corresponding decreases in the [Ca2+]i transient. Thus, inhibition of the ICa was reflected by a change in the peak [Ca2+]i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of [Ca2+]i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of [Ca2+]i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, [Ca2+]i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in [Ca2+]i. In contrast, [Ca2+]i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na(+)-dependent Ca2+ efflux did not significantly contribute to buffering [Ca2+]i transients in dorsal root ganglion neurone cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1990.sp018094

Stanley A. Thayer

Papers

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Cannabinoid Receptor Agonists Inhibit Glutamatergic Synaptic Transmission in Rat Hippocampal Cultures

Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons

Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons

Regulation of the intracellular free calcium concentration in single rat dorsal root ganglion neurones in vitro.