**5.1. Comparison of the levels of L-tetraiodothyronine (L-T4) and Ltriiodothyronine (L-T3) in subcellular fractions**

While serm levels of L-T4 (~ 41 ng/ml) and L-T3 (~ 0.7 ng/ml) were found consistent with the normal peripheral results, this assay system could not detect L-T4 in either synaptosomal or non-synaptic mitochondrial fractions. However, the L-T3 levels in synaptosomes (0.450.06 ng/mg synaptosomal protein), and non-synaptic mitochondria (1.440.12 ng/mg "Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 9

8 Thyroid Hormone

**in adult rat brain cerebral cortex** 

adult brain compared to developing brain.

levels of THs in adult rat brain cerebral cortex.

**triiodothyronine (L-T3) in subcellular fractions** 

**5.1. Comparison of the levels of L-tetraiodothyronine (L-T4) and L-**

While serm levels of L-T4 (~ 41 ng/ml) and L-T3 (~ 0.7 ng/ml) were found consistent with the normal peripheral results, this assay system could not detect L-T4 in either synaptosomal or non-synaptic mitochondrial fractions. However, the L-T3 levels in synaptosomes (0.450.06 ng/mg synaptosomal protein), and non-synaptic mitochondria (1.440.12 ng/mg

Synaptosomes are frequently used to study synaptic signal transduction pathways because they contain almost the entire molecular machinery necessary known for the uptake,

**5. Subcellular levels of L-triiodothyronine (L-T3) and L-thyroxine (L-T4)** 

As the brain approaches adulthood, nuclear iodothyronine concentrations gradually decreases reaching a plateau and maintains it, and the TH levels increase within nerve terminals of adult vertebrates [1,18-21]. It also demonstrated decrease in L-T3-binding in

Although, evidence of transportation 125I-L-T3 and 125I-L-T4 within the nerve terminal was demonstrated following intravenous injection in adult rat brain [10,18,19,22], its euthyroid concentrations and subcellular distribution was never been evaluated until recently [13,23]. Intravenous administration of [125I]-L-T4 in rats followed by thaw mount autoradiography showed distribution of L-T4 in selective areas of adult brain in a saturable manner. Gradually L-T4 was concentrated more within nerve terminals fractions, where L-T4 was monodeiodinated to produce L-T3, the active form of TH [10]. L-T4 and L-T3 transportation within neurons are shown to occur by two different mechanisms. L-T3 is actively taken up in a saturable manner, while L-T4 transportation occurs by diffusion and in a non-saturable way. L-T4-transporation within the neuron is dependent upon L-T4-concentration gradient between extracellular and intracellular compartments and is maintained by high deiodination rate of L-T4 to L-T3 [24]. Role of transthyretin has also been described as a major binding protein in cerebrospinal fluid. Transthyretin has been implicated to facilitate L-T4 transportation across the blood-brain-barrier and finally into the brain. Recently MCT-8 has been ascribed to be the most effective TH transporter [25]. These MCT-8 are 12 transmembrane spanning proteins, and in particular plays a major role for very specific transportation of L-T3 within the neurons followed by the active conversion of the prohormone L-T4 to L-T3 by the D-II within the CNS [26]. D-II is essentially important for the conversion of the prohormone L-T4 into the active L-T3 within the CNS. However, understandings of the levels of THs within the neurons are imperative. This information is crucial to explore the role of L-T3 in neural signal transmission in mature brain. To help meet this requirement the following study was performed to quantify and compare the

storage, release of neurotransmitters, receptor properties, and enzyme actions etc.

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 [29,30].

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

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 endogenously bound form of the hormones [13].

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

compared to euthyroid control values. A single i. p. injection of L-T3 (2 g/g BW) to the hypothyroid rats decreased the synaptosomal levels of L-T3 by ~1.6-fold compared to the hypothyroid rats and was still ~6-fold higher than the euthyroid value. An increase in ~2.5 fold of the L-T3 levels was noticed in euthyroid plus L-T3 (2 g/g BW) group (Figure 4) [13]. Although the levels of L-T3 in whole rat brain homogenate was found to be in low nanomolar ranges [22], two concurrent reports estimated synaptosomal levels of L-T3 to be ~14.6 nM [23], and ~13 nM [13] in adult rat brain synaptosomes. Observation of high levels of synaptosomal L-T3 were also supportive [15] in hypothyroid rat cerebral cortex by ~1.7 fold compared to the control values maximally at day 4 of induction of hypothyroidism while the serum levels of L-T3 remained at the hypothyroid levels.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 11

hypothyroid condition for a much longer duration as used by other workers. This may be one of the reasons for maintaining a high level of synaptosomal L-T3 in our hypothyroid rats. Expression of the data in different forms such as per gram organ (brain) basis, or per mg compartmental (synaptosomal) protein basis, as presented in our experiment, also becomes an additive factor for discrepancies among different groups of workers regarding the quantitative aspects of L-T3 or L-T4 in the brain [23,34,38,39]. The fall in L-T3 concentration in synaptosomes prepared from L-T3-treated hypothyroid rat cerebral cortex may be the result of inhibition of D-II activity after 24 hours of the L-T3 administration, in the presence of the considerable amount of exogenous L-T3. An inhibition in the activity of D-II has been noticed within 4 hours of L-T3 treatment to the thyroidectomized rats. A rise in the synaptosomal L-T3 level in the hypothyroid rats, and a fall in the same in the L-T3 treated hypothyroid animals after 24 hours of L-T3-treatment, also reflects the tendency for a compensatory regulatory mechanism of thyroid hormone metabolism in the adult rat brain in altered thyroid conditions, although the nature of the mechanism remains unknown. L-T3-treated control rats have shown higher levels of synaptosomal L-T3, compared to the control values. This may be a result of the extra L-T3 transport influenced

Observation of undetected levels of L-T4 within cerebrocortical synaptosomes may reflect a state of rapid conversion of L-T4 to L-T3 in the brain by D-II enzyme. Other researchers have already shown that after intravenous administration of radiolabeled L-T4 and L-T3, the hormone is concentrated as L-T3 in a synaptosomal fraction of the whole rat brain, and L-T4 to L-T3 conversion occurs very rapidly within the nerve cells. L-T3 formed in the neuronal cell body then may be translocated down the axon to the synaptic ends. Saturable and nonsaturable uptake of L-T3 and L-T4 in isolated synaptosomes in an *in vitro* model also

The prediction of a role of D-II as suggested [13] is further supported by few other studies [15,31]. Increased D-II activity is suggested in hypothyroid brain. This is attributed to the maintenance of normal brain concentrations of L-T3 even under low peripheral levels of L-T4 [31]. The high level of L-T3 as observed by us is supported and suggested for maintenance of brain homeostasis. This demonstrated onset of a central homeostasis for THs in adult hypothyroid brain between the 1st and 2nd day, its maintenance for about 16-18 days and thereafter declined between the 18-20th day [15]. This report also confirms and confers higher activity of D-II (~ 1.6-fold higher compared to control) within the cerebrocortical synaptosomal fraction during short-term brain-hypothyroidsm. It is described as a protective mechanism of brain by raising the brain L-T3 levels. Another study also documents an increase in D-II activity within various brain regions and decrease in D-III activity, except in cerebrellum and medulla where specific D-III activity remained undetected [40]. However, controversially, although these investigation did observe higher D-II activity within various areas of adult brain during hypothyroidism, the changes in L-T3 levels remained lower than normal values as was noticed in case of serum levels of hypothyroidism. This investigation could not explain this high D-II activity and lower L-T3 levels in brain regions. The levels of THs measured in this study also were shown to be

by a high dose of exogenously administered L-T3 (2 g/g) [18,19,24].

indicated two-component L-T3-uptake system [18,19,24,37,38].

Hypothyroid condition shows an appreciable decline in both serum L-T4 and L-T3 level in rats in a usual way as found by other investigators [34]. Although it has been shown earlier that in hypothyroid condition, the whole brain, or different regions of the brain, maintain similar levels of L-T3 compared to the euthyroid control rats through increased activity of D-II, and corresponding high fractional rate of L-T4 to L-T3 conversion [35,36], insufficient evidence is available except for a few recent reports to quantitate the synaptosomal concentration of thyroid hormones. Approximately 8-fold higher concentration of L-T3 has been found in synaptosome compared to the whole brain in euthyroid rats. Our observation of approximately 9.5-fold higher L-T3 content in synaptosome of hypothyroid rats compared to the euthyroid controls may be the result of a higher fractional rate of L-T3 production by increased activity of D-II, and a correspondingly higher selective uptake and concentration of L-T3 molecules in the synaptosomes to cope up with the physiological need of THs in this tissue at this condition [13,23,37,38].

**Figure 4.** L-T3 levels in rat cerebrocortical synaptosomes in various thyroid states. (Ref. Sarkar and Ray 1994, Neuropsychopharmacology 11: 151-155 acknowledged [13]).

In euthyroid rat brain, selective uptake of 125I-L-T3 and its concentration in synaptosomal compartment have been demonstrated [10]. In addition, the use of hypothyroid animals only after 14 days of PTU treatment, where some adaptive mechanisms still unknown in nature prevail, do not reach the equilibrium as compared to the animals kept in chronic

compared to euthyroid control values. A single i. p. injection of L-T3 (2 g/g BW) to the hypothyroid rats decreased the synaptosomal levels of L-T3 by ~1.6-fold compared to the hypothyroid rats and was still ~6-fold higher than the euthyroid value. An increase in ~2.5 fold of the L-T3 levels was noticed in euthyroid plus L-T3 (2 g/g BW) group (Figure 4) [13]. Although the levels of L-T3 in whole rat brain homogenate was found to be in low nanomolar ranges [22], two concurrent reports estimated synaptosomal levels of L-T3 to be ~14.6 nM [23], and ~13 nM [13] in adult rat brain synaptosomes. Observation of high levels of synaptosomal L-T3 were also supportive [15] in hypothyroid rat cerebral cortex by ~1.7 fold compared to the control values maximally at day 4 of induction of hypothyroidism

Hypothyroid condition shows an appreciable decline in both serum L-T4 and L-T3 level in rats in a usual way as found by other investigators [34]. Although it has been shown earlier that in hypothyroid condition, the whole brain, or different regions of the brain, maintain similar levels of L-T3 compared to the euthyroid control rats through increased activity of D-II, and corresponding high fractional rate of L-T4 to L-T3 conversion [35,36], insufficient evidence is available except for a few recent reports to quantitate the synaptosomal concentration of thyroid hormones. Approximately 8-fold higher concentration of L-T3 has been found in synaptosome compared to the whole brain in euthyroid rats. Our observation of approximately 9.5-fold higher L-T3 content in synaptosome of hypothyroid rats compared to the euthyroid controls may be the result of a higher fractional rate of L-T3 production by increased activity of D-II, and a correspondingly higher selective uptake and concentration of L-T3 molecules in the synaptosomes to cope up with the physiological need

**Figure 4.** L-T3 levels in rat cerebrocortical synaptosomes in various thyroid states. (Ref. Sarkar and Ray

Treatment Control Hypo Hypo + T3 T3

Synaptosomal L-T3 level

In euthyroid rat brain, selective uptake of 125I-L-T3 and its concentration in synaptosomal compartment have been demonstrated [10]. In addition, the use of hypothyroid animals only after 14 days of PTU treatment, where some adaptive mechanisms still unknown in nature prevail, do not reach the equilibrium as compared to the animals kept in chronic

while the serum levels of L-T3 remained at the hypothyroid levels.

of THs in this tissue at this condition [13,23,37,38].

L-T3 (ng/mg synaptosomal protein)

0

1

2

3

4

5

1994, Neuropsychopharmacology 11: 151-155 acknowledged [13]).

hypothyroid condition for a much longer duration as used by other workers. This may be one of the reasons for maintaining a high level of synaptosomal L-T3 in our hypothyroid rats. Expression of the data in different forms such as per gram organ (brain) basis, or per mg compartmental (synaptosomal) protein basis, as presented in our experiment, also becomes an additive factor for discrepancies among different groups of workers regarding the quantitative aspects of L-T3 or L-T4 in the brain [23,34,38,39]. The fall in L-T3 concentration in synaptosomes prepared from L-T3-treated hypothyroid rat cerebral cortex may be the result of inhibition of D-II activity after 24 hours of the L-T3 administration, in the presence of the considerable amount of exogenous L-T3. An inhibition in the activity of D-II has been noticed within 4 hours of L-T3 treatment to the thyroidectomized rats. A rise in the synaptosomal L-T3 level in the hypothyroid rats, and a fall in the same in the L-T3 treated hypothyroid animals after 24 hours of L-T3-treatment, also reflects the tendency for a compensatory regulatory mechanism of thyroid hormone metabolism in the adult rat brain in altered thyroid conditions, although the nature of the mechanism remains unknown. L-T3-treated control rats have shown higher levels of synaptosomal L-T3, compared to the control values. This may be a result of the extra L-T3 transport influenced by a high dose of exogenously administered L-T3 (2 g/g) [18,19,24].

Observation of undetected levels of L-T4 within cerebrocortical synaptosomes may reflect a state of rapid conversion of L-T4 to L-T3 in the brain by D-II enzyme. Other researchers have already shown that after intravenous administration of radiolabeled L-T4 and L-T3, the hormone is concentrated as L-T3 in a synaptosomal fraction of the whole rat brain, and L-T4 to L-T3 conversion occurs very rapidly within the nerve cells. L-T3 formed in the neuronal cell body then may be translocated down the axon to the synaptic ends. Saturable and nonsaturable uptake of L-T3 and L-T4 in isolated synaptosomes in an *in vitro* model also indicated two-component L-T3-uptake system [18,19,24,37,38].

The prediction of a role of D-II as suggested [13] is further supported by few other studies [15,31]. Increased D-II activity is suggested in hypothyroid brain. This is attributed to the maintenance of normal brain concentrations of L-T3 even under low peripheral levels of L-T4 [31]. The high level of L-T3 as observed by us is supported and suggested for maintenance of brain homeostasis. This demonstrated onset of a central homeostasis for THs in adult hypothyroid brain between the 1st and 2nd day, its maintenance for about 16-18 days and thereafter declined between the 18-20th day [15]. This report also confirms and confers higher activity of D-II (~ 1.6-fold higher compared to control) within the cerebrocortical synaptosomal fraction during short-term brain-hypothyroidsm. It is described as a protective mechanism of brain by raising the brain L-T3 levels. Another study also documents an increase in D-II activity within various brain regions and decrease in D-III activity, except in cerebrellum and medulla where specific D-III activity remained undetected [40]. However, controversially, although these investigation did observe higher D-II activity within various areas of adult brain during hypothyroidism, the changes in L-T3 levels remained lower than normal values as was noticed in case of serum levels of hypothyroidism. This investigation could not explain this high D-II activity and lower L-T3 levels in brain regions. The levels of THs measured in this study also were shown to be

lower than found by other investigators. Some assay in brain regions was also performed in tissue homogenates instead of particular subcellular fractions. Possibly differences in the concentrations of THs could be due to a different method of severe extraction procedure employed to extract brain tissue THs resulting in loss of it.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 13

(2g/g BW) to almost close to the euthyroid levels. However, this study could not distinguish between the genomic and nongenomic effects of L-T3. TH has also been reported to inuence K+-evoked release of [3H]-GABA in adult rat cerebrocortical synaptosomes. Such evidence indicates a possible role of TH in neurotransmission in adult mammalian brain. A functional correlation between L-T3 binding and the corresponding inhibition of NKA activity under *in vitro* conditions in the synaptosomes of adult rat cerebral cortex were established [46]. To further test the hypothesis of nongenomic action of TH we investigated NKA activity in isolated synaptosomes which is devoid of nucleus to avoid the chances of nuclear activation [46]. In fact, *in vitro* addition of L-T3 (1x10-12 M to 10x10-8 M) within 10 minutes of incubation indicated a dose-dependent inhibitory response to NKA activity. Such immediate action of L-T3 added in *in vitro* in synaptosomes was concluded as rapid nongenomic action of L-T3 on synaptosomal membrane NKA [46]. Further inhibition of NKA activity was corroborated with gradual binding of [125I]-L-T3 to specific L-T3-binding sites in synaptosomes. Thus a physiologic response tied to the specific

The presence of high affinity low capacity nuclear TH receptors in adult rat brain has been reported. Further evidence shows selective uptake of [125I]-L-T3 and rapid conversion of L-T4 to L-T3 in synaptosomal fraction of adult rat brain. Specic [125I]-L-T3 binding sites have also been demonstrated in the synaptosomes of adult rat brain [47] and chick embryo [48]. However, no functional relationship could be established due to the interaction of TH and

Scatchard plot analysis demonstrated two sets of specific L-T3 binding sites: one with high affinity (Kd1: 12 pM; Bmax1: 3.73±0.07 fmols/mg protein), and the other with low affinity (Kd2: 1.4±0.05 nM; Bmax2: 349±7 fmols/mg protein). Kd represents dissociation constant. Bmax represents maximum binding capacity. Rationale between gradual L-T3 binding and the corresponding dose-dependent L-T3-induced inhibition of synaptosomal NKA was

The relative order of potencies of binding afnities for the synaptosomal L-T3 binding sites and relative inhibition of NKA activity in the presence of different L-T3 analogues were as follows: L-T3>L-T3-amine>L-T4=L-TRIAC>r-T3>L-T2, and L-T3>L-T3-amine>L-T4>L-TRIAC>r-T3>L-T2, respectively. The concentrations of TH analogues required to displace 50% specic binding (ED50 value) of 125I-L-T3 to its synaptosomal binding sites were 10-, 63-, 63-, 1000- and 6250 nM, respectively. This study showed the nature of inhibition of synaptosomal NKA activity as a function of L-T3 occupancy of synaptosomal receptor sites

This investigation demonstrates a novel action of TH in mature rat brain. This is the rst report presenting a relationship between the inhibitions of synaptosomal NKA as a functional effect of L-T3 binding to its synaptosomal receptor in the cerebral cortex of adult rat. Occupancy of specic high afnity L-T3 binding sites demonstrated a concentrationdependent inhibition of the NKA activity with a maximum of 59%. At 1x10–10 M L-T3 concentration the enzyme inhibition was ~35% and the saturation of the L-T3 binding sites

L-T3-binding in the synaptosomal membrane was demonstrated.

its membrane receptor so far in adult brain.

established *in vitro* [46].

in mature rat brain [46].

The data emerged from our study reveal the quantitative aspects of involvement of L-T3 in synaptosomes in different thyroid states, and favors its role in neuronal functions as formerly described [10,41]. A stimulation of synthesis of synapsin-1 protein (related to neurotransmission) by L-T3 in the developing brain has been reported [42]. Although, the synaptosomal L-T3 levels varied widely with different treatments, our result illustrates a unique, but unknown regulatory mechanism of the TH metabolism in the mature mammalian brain.
