**4. Experimental tissue of interest**

Author's experiments and results reported in this manuscript are obtained from the purified synaptosomes prepared from young adult rat brain cerebral cortex. Synaptosomes are subcellular nucleus-free preparation purified through density gradient centrifugation [17]. The question may arise why synaptosome? Synapses are the ultimate routes of communications in neurons where electrical impulses are normally translated to chemical signals from one neuron to the other leading to subsequent biochemical and physiologic events. This preparation is a fragment of neurons containing the neuronal membrane, "Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 7

6 Thyroid Hormone

**2. Aim of the article** 

phosphorylation.

**3. Hypothesis** 

in the tanycytes lining the lower part of the third ventricles. D-III catalyzes the conversion of L-T3 to L-T2. Concentration of L-T3 within the nervous system has been attributed to the brain D-II which has major functions in regulating the overall neuronal homeostasis for TH. Expression of D-II in nervous tissue is implicated in the neuronal uptake of the circulatory L-T4 and its conversion to L-T3 followed by its supply to the neuronal targets. Expression of D-II is an important protective mechanism against hypothyroidism. This prevalence of TH

Interest also materializes to explore further the nongenomic mechanism of action of THs in adult mammalian CNS. In this context TH-mediated signal transduction pathways are also being investigated. Particularly the regulation of the activation of the second messenger systems and subsequent protein phosphorylation are of much awareness. Understanding of the mechanism of action of TH in adult mammalian brain has key implications in the higher mental functions, learning and memory, and in the regulation of several neuropsychiatric

The major goal of this article is to search, discuss and review the nongenomic rapid actions of THs in mature mammalian CNS. This article aims to begin with observations describing subcellular distribution, and concentrations of THs within the brain and its biochemical and physiologic consequences, specific binding of THs onto the neuronal plasma membrane to examine for specific plasma membrane receptors of THs and correlate the receptor-binding followed by a specific cellular function. Next, the molecular basis of the TH and plasma membrane receptor interaction-mediated signals are evaluated via possible activation of Gprotein signaling pathway, second messenger systems, and subsequent target protein

Thyroid hormones exercise a nongenomic action on the adult mammalian brain possibly by binding to neuronal membrane receptors followed by activation of second messenger cascade systems leading to substrate level protein phosphorylation and dephosphorylation

Author's experiments and results reported in this manuscript are obtained from the purified synaptosomes prepared from young adult rat brain cerebral cortex. Synaptosomes are subcellular nucleus-free preparation purified through density gradient centrifugation [17]. The question may arise why synaptosome? Synapses are the ultimate routes of communications in neurons where electrical impulses are normally translated to chemical signals from one neuron to the other leading to subsequent biochemical and physiologic events. This preparation is a fragment of neurons containing the neuronal membrane,

by protein kinases and protein phosphatases (Figure 2).

**4. Experimental tissue of interest** 

homeostasis is a preventive measure and thought to be neuroprotective [1,13-16].

disorders developed during adult-onset thyroid dysfunctions in humans.

**Figure 2.** Hypothesis: Proposed nongenomic action of thyroid hormones in adult mammalian brain.

**Figure 3.** (a) A typical neuron. (b) Cartoon of a neuron showing synaptosome. (c) Scanning electron microscopic image of synaptosome.

Synaptic vesicles, and the other intracellular components (Figure 3). Synaptosomes can be considered as isolated nerve terminals. Synaptosomes are obtained after homogenization and fractionation of nerve tissue. The fractionation step involves several centrifugations steps to separate various organelles from the synaptosomes. Synaptosomes are formed from the phospholipid layer of the cell membrane and synaptic proteins such as receptors.

Synaptosomes are frequently used to study synaptic signal transduction pathways because they contain almost the entire molecular machinery necessary known for the uptake, storage, release of neurotransmitters, receptor properties, and enzyme actions etc.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 9

mitochondrial protein) were significant. The levels of L-T3 in non-synaptic mitochondria were ~3.2-fold higher compared to synaptosomal values in cerebral cortices [13,16]. The finding of undetectable levels of synaptosomal L-T4 was consistent with other studies [14,27,28]. A higher fractional rate of D-II activity that converts L-T4 to L-T3 is attributed

This study quantifies the TH concentrations from adult rat brain synaptosomal and nonsynaptic mitochondria. Although L-T4 levels could not be detected in synaptosomal and nonsynaptic mitochondrial fractions, fair amounts of L-T3 were detected in these fractions purified from adult rat brain cerebral cortex [13,16]. Undetectable levels of synaptosomal L-T4 levels

Despite very low levels of TH in hypothyroid condition as determined by serum levels of TH, previous report has shown that L-T3 production in brain is pretty high in stress situations like hypothyroidism [13]. D-II has also been shown to be activated in other stressful conditions and indicated to have a protective role in stressed brain [31]. Stimulated levels of D-II have been described during hypothyroidism. This supports the first initial report [13] of elevation of brain L-T3 levels during n-propylthiouracil (PTU)-induced hypothyroid conditions [14,15,32]. In brain, approximately 80% of the L-T3 is produced locally from L-T4 by D-II. The fractional rate of conversion of L-T4 to L-T3 is remarkably high in brain [29]. This might be a possible reason for undetectable L-T4 levels due to rapid conversion of L-T4 to L-T3 in these fractions. To detect the endogenous TH levels the subcellular fractions were ruptured hypo-osmotically. The use of 8-anilinonaphtho-sulfonic acid in the radioimmunoassay medium excluded the possibility of the non-detectable protein bound form of the hormone by releasing the

Comparatively higher levels of L-T3 in the mitochondria may have implications on the mitochondrial bioenergetics such as, cellular oxygen consumption, oxidative phosphorylation and ATP synthesis, mitochondrial gene expression. These are few of the major regulatory functions of TH. THs also have been shown to affect mitochondrial genome mediated through imported isoforms of nuclear TH receptors and influence various mitochondrial transcription factors [3,33]. Concentration and localization of radiolabeled L-T3 within the nerve terminal was the first landmark research described in adult rat brain. This further followed with the immunohistochemical mapping demonstrating locus ceruleus norepinephrine stimulating active conversion of L-T4 to L-T3. This established a morphologic co-localization of central thyronergic and noradrenergic systems. Overall TH levels within different compartment of brain may have discrete, differential and potential

regulatory function for neurotransmission in adult mammalian brain [10].

*5.1.1. Thyroid hormone levels in hypothyroid rat cerebrocortical synaptosomes* 

Synaptosomal levels of L-T3 were also studied in different thyroidal conditions. Serum levels of L-T3 and L-T4 confirmed establishment of peripheral hypothyroidism induced by 14 days of intra-peritoneal (i. p.) injections of PTU (2 mg/g BW). However, surprisingly hypothyroid rat brain showed ~9.5-fold higher amount of L-T3 (126 nM) in synaptosomes

were also supported within synaptosomal fractions obtained from adult rat brain [27].

endogenously bound form of the hormones [13].

[29,30].
