science notes

by T. J. Nelson

No, they don't stand on chairs screaming when one of us gets out of our cage. Not anymore. Female humans don't make distress calls like they used to. But male and female brains do have many innate differences, not just in math and spatial processing, but also in connectivity. This week we learned something really earth-shattering: the wiring for pain in male and females is different. At least for us mice.

Top: Minocycline, Bottom: Tetracycline

To understand this result, it's necessary to understand what we mouse researchers did before.

We've known for a long time that the VTA (ventral tegmental area), a group of dopamine-secreting neurons in the midbrain, is involved in the reward circuitry of the brain. Dopamine acts as an analgesic signal and as the reward signal in the VTA. That means dopamine is involved in sexual orgasm and motivation, but also addiction and chronic pain. The VTA is next to the substantia nigra, which is another set of dopaminergic neurons, that is affected in Parkinson's disease.

Antibiotics that can cross the blood-brain barrier and get into the brain, such as minocycline, inhibit microglial cells. So Taylor et al. [1] used minocycline to probe the biochemical mechanism. They found:

• Chronic pain decreases the amount of dopamine (reward signal) released from the VTA, while opioids (like morphine) and cocaine increase it.
• Chronic pain interferes with morphine's ability to affect the reward signal, but does not interfere with its ability to block nociception (pain signals).
• Activating microglia blocks the release of dopamine.
• Inhibiting microglia with minocycline restores the dopamine signal.

The conclusion is that chronic pain is mediated by loss of dopamine release in the VTA. This is apparently caused by activation of microglia.

This is big. Microglia are the ‘immune cells’ of the brain. Their purpose is to clean up debris in the brain. They mediate chronic inflammation, which occurs in Alzheimer's disease. They also chew up and destroy synapses that are injured.

(It's necessary to add that these mice weren't suffering in this experiment. In fact, with all the drugs they were given, they were probably pretty happy about the whole thing.)

These microglia also produce a lot of BDNF (brain-derived neurotrophic factor), which causes neurons to grow and form new synapses. It's involved in learning, and it's a very important protein in the brain. But it turns out that BDNF also strengthens the neural circuits that block the dopamine reward signal.

Synapses are good, because they're needed for memory. But too many synapses can be as bad as too few. In some forms of autism, a disease that affects males 2−3 times more than females, there are defects in synaptic maturation. In others, there are too many synapses. Microglia are suspected here [2]. Some researchers also suspect that in Rett syndrome, a rare genetic form of autism that affects only girls, may also be related to suppression of microglial neuroinflammation [3]. We're only starting to understand the important role played by the brain's immune system.

Normally, activating microglia only produces a mild anti-inflammatory response, called M2 activation. But when sufficiently provoked, microglia can turn nasty. In their pro-inflammatory response, called M1 activation, they produce reactive oxygen species (such as superoxide radicals), NOS (an enzyme that produces the gas nitric oxide), and inflammatory cytokines that can kill bacteria and even neurons. So we need to keep an eye on those microglia.

Humans in the mist

Now a bunch of Canadian and American researchers, publishing in Nature Neuroscience [4], have blown all that out of the water. Female mice don't use microglia at all for chronic pain hypersensitivity (called allodynia). They use T lymphocytes instead.

Sorge et al. discovered that minocycline, the microglia inhibitor, doesn't do a darn thing in female mice. Since this is a negative finding, most of their short article is devoted to making sure nothing went wrong. They tried other ways of getting rid of microglia. Same result. They created a transgenic strain with no BDNF in the microglia. It wiped out the allodynia in males, but not in the females. Yet the amount of allodynia in untreated wild-type mice was the same.

Female mice, of course, still have microglia. On a hunch Sorge et al. went out and bought some female mice with no T-cells. They turned out to be using the microglia pathway just like the males. When they put the T-cells back (using adoptive splenocyte transfer), they became insensitive to minocycline again. This means that in females, the T-cells are adapting and taking over the function of microglia. Fenofibrate, a drug that activates a protein called PPARα (peroxisome proliferator activated receptor alpha), wiped out the allodynia response in males, but not females. Conversely, a PPARγ agonist (pioglitazone) wiped it out in females, but not in males. Expression of these PPAR proteins in T-cells is sexually dimorphic, which means it's influenced by testosterone. So there ya go. A reasonable explanation.

There's good reason to believe these results apply to you humans as well. Minocycline penetrates the CNS more readily than other antibiotics like tetracycline. It produces vestibular side effects that are much more common in women than in men [5].

This paper illustrates the beauty of science. A discovery starts when you look at something a little different and an unexpected result happens. Finding out why the numbers didn't come out right leads, if the pieces are in place, to the solution of a previously unsolved mystery. Just remember: you couldn't have done it without us mice.

You humans don't use female mice much in research, but it's not because you're sexist. You use them, I'm told, because female mice are a b**** to work with because of their estrous cycle. (Tell me about it.) So it turns out that Tim Hunt was right about girls in the lab. But now, for brain research, you have no choice but to use them, and to make them cry, or at least squeak, some more.

References

[1] J. Neurosci. 2015 Jun 3;35(22):8442-50. Microglia disrupt mesolimbic reward circuitry in chronic pain. Taylor AM, Castonguay A, Taylor AJ, Murphy NP, Ghogha A, Cook C, Xue L, Olmstead MC, De Koninck Y, Evans CJ, Cahill CM.

[2] Dev Neurosci. 2015;37(3):195-202. Role of Microglia in Autism: Recent Advances. Takano T.

[3] Neuron Glia Biol. 2011 Feb;7(1):85-97. Does microglial dysfunction play a role in autism and Rett syndrome? Maezawa I, Calafiore M, Wulff H, Jin LW.

[4] Different immune cells mediate mechanical pain hypersensitivity in male and female mice, Sorge RE, Mapplebeck JC, Rosen S, Beggs S, Taves S, Alexander JK, Martin LJ, Austin JS, Sotocinal SG, Chen D, Yang M, Shi XQ, Huang H, Pillon NJ, Bilan PJ, Tu Y, Klip A, Ji RR, Zhang J, Salter MW, Mogil JS. Nat Neurosci. 2015 Jun 29. article abstract

[5] Antimicrob Agents Chemother. 1977 Apr;11(4):712-7. Side effects of minocycline: a double-blind study. Fanning WL, Gump DW, Sofferman RA.