Re: More evidence of inflammation and depression.

Minocycline protects neurons from dying by suppressing toxic events. Cell death triggers immune resposes that include brain inflammation. One mechanism by which minocycline protects these cells is the suppression of glutamatergic hyperexcitability and Ca2+ ion influx. Minocycline also protects cells by reducing the formation of damaging oxidative free radicals inside the cell. These species can wreak havoc with events inside the cell nucleus. Minocycline thus reduces the damage to cell viability by suppressing glutamate overacitiviy.

Of particular interest to me is the ability of minocycline to inhibit the release of glutamate. I believe that this effect works synergistically with the antiglutamatergic effects of Lamictal. Combining these two drugs together might prove especially effective in the treatment of depression, particularly when there is a bipolar diathesis.

Unlike the dogma promoted in decades gone by that supported the notion that affective disorders were not organic, we now realize that mood illnesses are degenerative. Cells die. Connections die. Fortunately, when one responds well to drug treatment, many of these cells recover while new cells are being born. The hippocampus is a brain structure known to facilitate memory and modulate mood. It shrinks (atrophy?) with depression. The recovery of this structure is easily measured when antidepressants are administered.

Minocycline reminds me of lithium. It has many, many different biological properties that seem to converge to produce its therapeutic effects.

I spent some time trying to excise passages from the following text in various ways, but I found that I kept deleting important information. So, I kept most of it and posted it here. It is long and somewhat technical, but I skipped the methods and results sections and their nomenclature jibberish. Just skim and skip to the good parts. I still must skim long texts due to my impaired concentration. I am not very happy with how long it is taking me to regain my abilility to read with my current treatment.

Neuroprotectant minocycline depresses glutamatergic neurotransmission and Ca2+ signalling in hippocampal neurons

José Carlos González,1,2 Javier Egea,1,2 María del Carmen Godino,3 Francisco J. Fernandez-Gomez,5
José Sánchez-Prieto,3 Luís Gandía,1,2 Antonio G. García,1,2,4 Joaquín Jordán5 and Jesús M. Hernández-Guijo1,2 1Instituto Teófilo Hernando, and
2Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain
3Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain
4Servicio de Farmacología Clínica, Hospital Universitario de la Princesa, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain
5Grupo de Neurofarmacología, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha y Centro Regional de Investigaciones Biomédicas, Albacete, Spain

The mechanism of the neuroprotective action of the tetracycline antibiotic minocycline against various neuron insults is controversial.
In an attempt to clarify this mechanism, we have studied here its effects on various electrophysiological parameters, Ca2+ signalling,
and glutamate release, in primary cultures of rat hippocampal neurons, and in synaptosomes. Spontaneous excitatory postsynaptic
currents and action potential firing were drastically decreased by minocycline at concentrations known to afford neuroprotection. The
drug also blocked whole-cell inward Na+ currents (INa) by 20%, and the whole-cell Ca2+ current (ICa) by about 30%. Minocycline
inhibited glutamate-evoked elevation of the cytosolic Ca2+ concentration ([Ca2+]c) by nearly 40%, and K+-evoked glutamate release
from synaptosomes by 63%. Minocycline also depressed the frequency and amplitude of spontaneous excitatory postsynaptic
currents, but did not affect the whole-cell inward current elicited by c-aminobutyric acid or glutamate. This pharmacological profile
suggests that the neuroprotective effects of minocycline might be associated with the mitigation of neuronal excitability, glutamate
release, and Ca2+ overloading.

The main aim of this study was to obtain better knowledge about the mechanism responsible for the neuroprotective effects of minocycline on glutamate-induced cytotoxicity. We used hippocampal neurons in culture, which are formed by approximately 80% of glutamatergic neurons (pyramidal cells) vs. 20% of GABAergic neurons (interneu- rons and granular cells); similar rates has been described in cortex primary culture(Millán et al.,2003). As shown in Fig.1, the glutamate treatment produced a significant decrease in cell viability.

To determine the effect of minocycline on glutamate-induced toxicity, hippocampal neurons were pretreated with a wide range of concen- trations (10-150 lm), which were observed to afford cytoprotection to the neuronal hippocampal culture in a concentration-dependent manner. We found in this study that neuroprotectant concentrations of minocycline against brain ischaemia (Yrjanheikki et al., 1999), excitotoxicity (Tikka et al., 2001), spinal cord injury (Stirling et al., 2004) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (He et al., 2001) depressed synaptic transmission in cultured hippocampal neurons. This depression is explained by reduction of sEPSCs, recorded in the presence of the GABAA receptor blocker bicuculline. The current frequency, the amplitude and the area of individual events are reduced approximately by 50% (Fig. 2). We demonstrate how minocycline could contribute to decrease neuronal excitability, not only by blocking depolarizing ionic channels, but also by an additional direct modulation of glutamate release. Hence, minocycline was probably reducing glutamate release, a mechanism that was directly tested in cortical synaptosomes, using high K+ as a depolarizing stimulus (Fig. 10). A postsynaptic effect explaining sEPSC depression is discarded, considering that minocycline did not affect either glutamate- or GABA-induced inward currents (Fig. 11). The presynaptic action of minocycline was corroborated by recording the spontaneous AP firing. Thus, the drug reduced the frequency of spontaneous APs recorded in current-clamped hippo- campal neurons (Fig. 3). Furthermore, the drug diminished the amplitude and the frequency of AP trains elicited by current injection, leading to a late suppression of cell firing (Fig. 4). The reduction of AP frequency is probably due to an increase in the after-hyperpolarisation duration, a slight increment of baseline, and a drastic increment in AP decay time. These changes augment the refractory period, and the subsequent decrease, of AP frequency. This effect of minocycline on AP generation and propagation may be explained by a combination of effects on various ion channel currents. For instance, minocycline reduced INa by 20% (Fig. 5), ICa by 30% (Fig. 8), and IKCa by 50% (Fig. 7). We measured the contribution of Ca2+-channels and voltage- dependent K+-channels to the total outward K+ current at potentials occurring during an AP, i.e. from )20 mV to +30 mV. This implies that over 22% of the K+ current measured in the first millisecond of a depolarizing step is activated by Ca2+ influx. Thus, blockade by minocycline of ICa (Fig. 8) may explain its blocking effect on IKCa (Fig. 7). Additionally, the possibility of Ca2+-dependent K+ current suppression exerted by a direct action of minocycline on Ca2+- dependent K+-channels is completely excluded by the similar IC50 obtained in both experimental approaches, blockade of ICa and IKCa. The Ca2+ currents were recorded in 10 mm external Ca2+ however, the use of high Ca2+ only shifted I / V 10 mV to the right (the control and the blockade curve). We performed some control experiments, recording the blockade exerted by minocycline at two potentials (0 and +10 mV) in comparison to 2 and 10 mm Ca2+, and no difference was detected. The extracellular concentration of Ca2+ did not affect the relative blockade exerted by minocycline. The blockade obtained in 10 mm Ca2+ at +10 mV is the same as that observed at 0 mV in 2 mm Ca2+. It was interesting that blockade was higher at test potentials (i.e. 0 mV), where ICa is known to be maximal; this corroborates the well- known observation that Ca2+ entering through voltage-dependent Ca2+-channels rapidly activates small- and large-conductance Ca2+- dependent K+-channels. As these channels control the post-hyperpo- larization phase of the AP and hence AP firing frequency [see Stocker (2004) for a review], the halving by minocycline of IKCa (Fig. 7) may explain the reduction of AP frequency and even the suppression of AP firing (Figs 3 and 4). The increment in the intracellular concentration of Ca2+ during neuronal ischaemia evoked by glutamate-derived hyperexcitability plays a particularly important role in the neurotoxic cascade resulting in acute neuronal cell death. Additionally, a reduction in the Ca2+ influx leads first to a decrement in cytosolic Ca2+ level to initiate the exocytotic process, and glutamate release, and second, to a decrement in the Ca2+-induced Ca2+ release responsible for maintenance of

Fig. 11. Effects of minocycline on glutamate- or c-aminobutyric acid (GABA)-evoked currents. (A) Hippocampal neurons in culture were perfused with Tyrode control solution and stimulated with 300 lm glutamate for 100 ms at 30-s intervals in the absence and presence of minocycline (added 2 min before). The histogram represents the average data for glutamate-induced responses in both conditions. (B) A hippocampal neuron perfused with Tyrode solution was stimulated with 100 lm GABA for 100 ms at 30-s intervals before (control trace) and during minocycline perfusion. The histogram represents the average data for GABA-induced responses in control conditions and in the presence of minocycline (added 2 min before). For each cell, the response in the presence of minocycline was calculated as a percentage of the response in control conditions (100%). Data are means ± SEM of number of neurons tested (indicated in parentheses). No statistical differences were found

Fig. 10. Minocycline reduces glutamate release in a concentration-dependent manner in cerebrocortical nerve terminals. (A) The release of glutamate evoked by 30 mm KCl in the presence of 1.33 mm Ca2+ or 5 mm EGTA was determined in the presence and absence of minocycline (10, 50 and 100 lm) added 100 s before depolarization. Note the lack of effect on external Ca2+- independent glutamate release (EGTA traces). Traces are the means of three to five experiments using two synaptosome preparations. (B) Averaged data of glutamate release in the absence (control) and the presence of minocycline. Data are means ± SEM of the number of cells shown in parentheses. **P < 0.01, with respect to control. (C) Minocycline fails to affect ionomy- cin-induced glutamate release. The lower trace shows the basal spontaneous release obtained. Glutamate release was induced by 6 lm ionomycin in the absence (control) or the presence of minocycline (100 lm), added 100 s before. Traces are the means of three to five experiments using two synaptosome preparations between the different experimental groups.

Vesicular transport for neurotransmitter release. The partial blockades exerted by minocycline of INa and ICa were translated into a 30-40% decrement of [Ca2+]c elevations elicited by glutamate (Fig. 9). This may explain the 60% blockade of K+-evoked glutamate release (Fig. 10). The K+-evoked release is not affected by either Na+-channel or K+-channel blockers, but is sensitive to inhibition by Ca2+-channel blockers (Millán et al., 2002). Nevertheless, the possibility of direct interference of minocycline with the exocytotic machinery release itself, downstream of Ca2+ entry, was excluded by the observation that minocycline did not affect the ionomycin-induced release of glutamate. Thus, these combined effects of minocycline may consid- erably reduce the neuronal Ca2+ overload evoked by excess glutamate stimulation of N-methyl-d-aspartate receptors, occurring during brain ischaemic insults (Siesjo et al., 1995). Observation of the effects of minocycline with the different experimental approaches suggested that 30 lm was the threshold concentration to exert all these effects. The effectiveness of minocycline achieved with the same concentration range (see similar calculated IC50) suggests that the blockade exerted on cytosolic Ca2+ elevation, and neurotransmitter release, which eventually led to a decrement in neuronal excitability, are mainly evoked by a direct blockade of voltage-dependent Ca2+-channels, without discounting a small contribution of other mechanisms, such as Na+-channel blockade. We have not considered an effect of minocycline on the different cell types and the possibility that pooling data from all cells may mask cell-type-specific effects because: (i) during the experiment performed, the hippocampal neurons recorded showed a pyramidal-like shape; (ii) the homogeneous effect recorded in every experimental approach, as shown by the low SEM, indicates a normal distribution effect, which excludes the possibility that different effects exerted by minocycline could be associated with different cell populations; and (iii) to prevent variations in the development of the primary culture, we employed 11-15-day-old neurons. Minocycline exhibits neuroprotective effects against neuronal damage in animal models of focal and global brain ischaemia (Yrjanheikki et al., 1999; Wang et al., 2003), Huntingtons disease (Chen et al., 2000; Wang et al., 2003), amyotrophic lateral sclerosis (Zhu et al., 2002), Alzheimers disease (Hunter et al., 2004), and Parkinsons disease (He et al., 2001). In particular, during and after ischaemic insults, there is consensus that excess glutamate release and impairment of glutamate sequestration by astrocytes might be the cause of exacerbated neurotoxicity and neuronal death [see Block et al. (2007) for a review]. In fact, therapeutic targets to mitigate such neurotoxicity include Ca2+-channel blockers to reduce excess gluta- mate release (Gribkoff & Winquist, 2005) or glutamate receptor blockers (García de Arriba et al., 2006). The cell viability experiments in which minocycline exerted a important neuroprotective effect, in contrast to the smaller effect evoked by other compounds, such as tetracycline, are in accordance with other studies showing that minocycline and doxycycline markedly reduce the size of infarction in both focal and global transient ischaemia in the adult rat (Clark et al., 1994; Yrjanheikki et al., 1999; Xu et al., 2004); by contrast, tetracycline, which is less able to cross the blood-brain barrier to enter the central nervous system, is not neuroprotective at the same doses (Yrjanheikki et al., 1998). The data available on the mechanism responsible for the neuropro- tective actions of minocycline are scarce and controversial (Jordán et al., 2007). Several reports attribute the minocycline neuroprotective effects to various intracellular signalling pathways, including antiox- idant systems (Kraus et al., 2005), nitric oxide synthase (Sadowski & Steinmeyer, 2001) and blockade of inflammatory responses [see Stirling et al. (2005) for a review]. Our results, however, strongly suggest that minocycline acts at an earlier plasmalemmal step by limiting glutamate release and the ensuing [Ca2+]c elevation in target neurons. Minocycline may prevent the activation of this Ca2+- dependent intracellular pathway, thus preventing neuronal death. The regulatory mechanism exerted by minocycline on Ca2+ entry and membrane potential leads to down-modulation of synaptic transmis- sion. This decrement in neuronal excitability, together with the marked decrement in glutamate release, may explain the cytoprotective properties of this drug. On the other hand, downregulation of the neuronal network activity may prevent microglial overactivation, with a favourable effect on neurodegenerative diseases. In fact, minocycline has been demonstrated to inhibit microglial activation (Yrjanheikki et al., 1999; He et al., 2001; Tikka et al., 2001), a finding that is in accordance with its neuroprotective effects in animal models of neurodegenerative diseases or ischaemia (Yrjanheikki et al., 1999; Chen et al., 2000; Zhu et al., 2002; Wang et al., 2003; Hunter et al., 2004). In conclusion, our observation that minocycline mitigates the excitability of hippocampal neurons by direct blocking of ionic channels involved in the generation and propagation of APs, and depresses glutamate release and Ca2+ overloading by the partial blockade exerted on voltage-dependent Ca2+-channels, may explain the well-studied neuroprotective properties of this tetracycline derivative in various in vitro and in vivo models of neurotoxicity.

Some see things as they are and ask why.
I dream of things that never were and ask why not.

- George Bernard Shaw

Re: More evidence of inflammation and depression.

Thread

Post a new follow-up

Notify the administrators

Start a new thread