Regulatory Considerations for the Clinical and Research Use of Transcranial Direct Current Stimulation (tDCS): review and recommendations from an expert panel.

The field of transcranial electrical stimulation (tES) has experienced significant growth in the past 15 years. One of the tES techniques leading this increased interest is transcranial direct current stimulation (tDCS). Significant research efforts have been devoted to determining the clinical potential of tDCS in humans. Despite the promising results obtained with tDCS in basic and clinical neuroscience, further progress has been impeded by a lack of clarity on international regulatory pathways. We therefore convened a group of research and clinician experts on tDCS to review the research and clinical use of tDCS. In this report, we review the regulatory status of tDCS, and we summarize the results according to research, off-label and compassionate use of tDCS in the following countries: Australia, Brazil, France, Germany, India, Iran, Italy, Portugal, South Korea, Taiwan and United States. Research use, off label treatment and compassionate use of tDCS are employed in most of the countries reviewed in this study. It is critical that a global or local effort is organized to pursue definite evidence to either approve and regulate or restrict the use of tDCS in clinical practice on the basis of adequate randomized controlled treatment trials.

Recombinant GABA(C) receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human rho1 subunit.

1. A recombinant adenovirus was generated with the human rho1 GABA(C) receptor subunit (adeno-rho). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant rho1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2. Standard whole-cell recording and Western blot analysis using rho1 GABA(C) receptor antibodies revealed that recombinant rho1 receptors were expressed in HEK293 and 911 cells after adeno-rho infection and exhibited properties similar to those of rho1 receptors after standard transfection. 3. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABA(C)-like current. After adeno-rho infection, however, a GABA(C)-like current appeared in 70-90 % of the neurons. 4. Five days after infection, expression of GABA(C) receptors in hippocampal neurons significantly decreased native GABA(A) receptor currents from 1200 +/- 300 to 150 +/- 70 pA (n = 10). The native glutamate-activated current was unchanged. 5. Hippocampal slices (P8) did not show a native GABA(C)-like current, although recombinant rho1 receptors could be expressed in cultured hippocampal slices after adeno-rho infection. 6. These data indicate that an adenovirus can be used to express recombinant GABA(C) receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases.

Protein synthesis-dependent and -independent regulation of hippocampal synapses by brain-derived neurotrophic factor.

A fundamental difference between short-term and long-term forms of synaptic plasticity is the dependence on transcription and translation of new genes. Using organotypic cultures of hippocampal slices, we have investigated whether the modulation of synapses by brain-derived neurotrophic factor (BDNF) also requires protein synthesis. Long-term treatment of hippocampal slice cultures with BDNF increased the number of docked vesicles, but not that of reserve pool vesicles, at CA1 excitatory synapses. BDNF also increased the levels of the vesicle proteins synaptophysin, synaptobrevin, and synaptotagmin, without affecting the presynaptic membrane proteins syntaxin and SNAP-25, or the vesicle-binding protein synapsin-I. The increase in synaptophysin and synaptobrevin expression was moderate (2-fold) and occurred within 6 h after BDNF application. In contrast, synaptotagmin expression took 24 h to reach maximum levels (5-fold). The delayed increase in synaptotagmin was blocked by protein synthesis inhibitors, while the early increase in synaptophysin and synaptobrevin was not. Moreover, the BDNF-induced increase of synaptotagmin was blocked by inhibiting the cAMP/protein kinase A (PKA) pathway. However, BDNF did not activate PKA, and application of a PKA activator did not mimic the BDNF effect. Taken together, these results suggest a novel, protein synthesis-dependent form of BDNF modulation that requires cAMP gating.

BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses.

Brain-derived neurotrophic factor (BDNF) is emerging as a key mediator of activity-dependent modifications of synaptic strength in the CNS. We investigated the hypothesis that BDNF enhances quantal neurotransmitter release by modulating the distribution of synaptic vesicles within presynaptic terminals using organotypic slice cultures of postnatal rat hippocampus. BDNF specifically increased the number of docked vesicles at the active zone of excitatory synapses on CA1 dendritic spines, with only a small increase in active zone size. In agreement with the hypothesis that an increased docked vesicle density enhances quantal neurotransmitter release, BDNF increased the frequency, but not the amplitude, of AMPA receptor-mediated miniature EPSCs (mEPSCs) recorded from CA1 pyramidal neurons in hippocampal slices. Synapse number, independently estimated from dendritic spine density and electron microscopy measurements, was also increased after BDNF treatment, indicating that the actions of BNDF on mEPSC frequency can be partially attributed to an increased synaptic density. Our results further suggest that all these actions were mediated via tyrosine kinase B (TrkB) receptor activation, established by inhibition of plasma membrane tyrosine kinases with K-252a. These results provide additional evidence of a fundamental role of the BDNF-TrkB signaling cascade in synaptic transmission, as well as in cellular models of hippocampus-dependent learning and memory.

Celiac vagotomy reduces suppression of feeding by jejunal fatty acid infusions.

We investigated the role of the celiac branch of the vagus nerve in suppression of food intake produced by jejunal fatty acids infusions. Following selective celiac vagotomy or sham surgery, adult, male Sprague-Dawley rats received 7 h infusions of linoleic acid or saline through indwelling jejunal catheters on four consecutive days. Although linoleic acid still produced significant suppression of intake in rats with celiac vagotomy, it was less effective in these animals than in controls. The temporal pattern of results suggested that celiac afferent fibers are involved in mediating both pre- and postabsorptive effects of infused fatty acids.

Suppression of food intake, body weight, and body fat by jejunal fatty acid infusions.

Three experiments investigated effects of jejunal lipid infusions given on 4 or 21 consecutive days in adult, male Sprague-Dawley rats. In experiment 1, 7-h infusions of linoleic or oleic acid (0.2 ml/h for 7 h; total load = 11.5 kcal) on 4 consecutive days reduced total intake (ad libitum consumption of the liquid diet Boost, Mead Johnson, plus load) by approximately 15% and decreased weight gain compared with 4-day tests with saline administration. In experiment 2, linoleic acid at 0.1 ml/h for 7 h (5.7 kcal) was ineffective, whereas the same load delivered in 3.5 h produced effects similar in magnitude to those in the first experiment. In experiment 3, jejunal infusions of linoleic acid (0.2 ml/h for 7 h) on 21 consecutive days reduced mean total intake by 16%, body weight by 10%, and carcass fat by 48% compared with controls receiving saline. The net decrease in caloric intake may reflect the combined activation of pre- and postabsorptive mechanisms, and it suggests a possible treatment for obesity.

Responses of celiac and cervical vagal afferents to infusions of lipids in the jejunum or ileum of the rat.

Multiunit celiac and single-unit cervical recordings of vagal afferents were performed before and during infusions of fatty acids, triglycerides, or saline into either the ileum or jejunum of the rat. In multiunit recordings, lipids increased activity of vagal afferents to a greater extent than saline. The greatest increases in vagal afferent activity resulted from infusions of linoleic acid, conjugated linoleic acid, or oleic acid. The triglycerides, corn oil or Intralipid, were less effective than the fatty acids in affecting vagal afferent activity. Ileal pretreatment with the hydrophobic surfactant Pluronic L-81 significantly attenuated the response of celiac vagal afferents to ileal infusion of linoleic acid. Single-unit recordings of cervical vagal afferents supported the multiunit data in showing lipid-induced increased vagal afferent activity in approximately 50% of ileal units sampled and 100% of a limited number of jejunal units sampled. These data demonstrate that free fatty acids can activate ileal and jejunal vagal afferents in the rat, and this effect can be attenuated by pretreatment with a chylomicron inhibitor. These data are consistent with the view that lipid-induced activation of vagal afferents could be a potential substrate for the inhibitory effects of intestinal lipids on gastrointestinal function, food intake, and body weight gain.

Inhibition of sucrose intake by continuous celiac, superior mesenteric, and intravenous CCK-8 infusions.

Two experiments compared the potency of continuous infusions of cholecystokinin octapeptide (CCK-8) for reducing sucrose intake when administered into abdominal arteries or the jugular vein. Adult, male Sprague-Dawley rats received 22-min infusions of saline or several doses of CCK-8. Sucrose was available for 20 min, beginning 2 min after onset of infusions. In the first experiment, intraceliac CCK-8 in doses of 50, 125, and 312 ng produced significant reductions in intake, but no dose affected intake when administered into the jugular vein. In experiment 2, only the highest dose, 312 ng, suppressed intake when infused into the superior mesenteric artery, and jugular infusions were again ineffective. Behavioral observations indicated that intra-arterial CCK-8 had no affect on feeding within the first several minutes of test meals but accelerated the subsequent decline in incidence of feeding. These results suggest that receptors involved in cholecystokinin satiety are widely distributed within the gastrointestinal tract.

Suppression of sucrose intake by continuous near-celiac and intravenous cholecystokinin infusions in rats.

We compared suppression of intake of 30% sucrose produced by continuous aortal (near celiac) and intravenous infusions of cholecystokinin octapeptide (CCK-8). Adult, male Sprague-Dawley rats received 21-min infusions of saline or 100-1,600 ng CCK-8. Sucrose was available for 20 min, beginning 1 min after onset of infusions. Significant reductions in intake were produced by near-celiac infusions of 400, 800, and 1,600 ng CCK-8, but only the two highest doses affected intake when infused intravenously. In a second experiment, which replicated previous observations, near-celiac bolus infusions of 70 ng CCK-8 significantly reduced sucrose intake but intravenous infusions did not. Behavioral observations indicated that although bolus infusions produced immediate disruption of feeding, suggesting an aversive effect, effects of continuous CCK-8 infusions on temporal intake patterns were consistent with enhancement of satiety. These data provide further evidence that CCK-8 acts on a site within the upper gastrointestinal tract to produce satiety.

Reproductive performance in genetic lines selected for high or average milk yield.

Genetic lines were created by selection of service sires differing by approximately 450 kg of milk for estimated transmitting ability. High line sires were selected from the best available proven sires. Selection continued over 24 yr with up to eight generations of selection. Records from 708 nulliparous, 575 first parity, and 437 second parity animals were analyzed. High milk yield was associated with longer days open and calving intervals in both first and second parities. A 1000-kg increase in 305-d milk production was associated with average increases in both days open and calving interval of around 7 d in first parity and 13 d in second parity and with average increases in days to first detected estrus of 4.5 d in first parity. Difference between genetic lines for milk yield was 804 kg in first parity and 772 kg in second parity. Days open and calving interval were less for the average line in both parities and differed by 10 d in second parity. Other reproductive differences were small or insignificant. Selection for yield has affected reproductive fitness modestly.

Influence of dietary protein concentration on milk production by dairy cattle during early lactation.

In a 3 X 2 factorial experiment 75 Holstein cows in first, second, or third lactation were fed rations containing either 12.2% or 16.2% crude protein in total ration dry matter. On the average, 26% of dry matter intake was from corn silage, 22% from alfalfa-grass hay, and 52% from a grain mix. Protein was controlled by feeding a 13.7% crude protein grain mix with 1.4% urea for the 12% ration and a 19.8% crude protein grain mix with natural protein for the 16% ration. Average daily milk production (kg/day) for wk 2 through 12 of lactation for 12% and 16% rations by lactations were: first, 21.6 and 21.9; second, 25.7 and 31.5; and third, 27.5 and 34.0. Dry matter intakes by lactations were .42, 1.18, and 2.05 kg/day higher for cows fed the high protein compared to low protein rations. Milk composition was not influenced by protein treatment. The markedly different response to protein supplementation in milk production between heifers in first lactation and more mature cows is unexplained.

Comparison of radioimmunoassay and gas-liquid chromatography analyses of progesterone concentrations in cow's milk.

Progesterone concentrations in milk were not significantly different when quantitated by different methods (RIA vs. GLC). There was a significant day effect (P less than 0.05) on milk progesterone level due apparently to gradually decreasing values as pregnancy advanced over days 30, 120 and 210. The means for the percent fat content were different (P less than 0.05) for all comparisons among four times of collection (immediately premilking, milking pool, immediately postmilking, and 3 hr postmilking). For progesterone concentration, the main effect of time and the three-way interaction (time times antiserum times purification method) were significant (P less than 0.005); all other main effects and interactions were not significant. Within each of 4 assay groups (2 antisera times 2 purifications), the concentration of progesterone was greater (P less than 0.05) for the samples which were collected immediately postmilking than for any of the other times of collection. The three-way interaction seemed due primarily to difference in progesterone determinations among the four assay groups in the samples which were taken immediately postmilking. Over all milk samples within each assay group, the percent fat content and the concentration of progesterone were positively correlated (r = 0.71, P less than 0.01).