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c-Fos expression mediated by N-methyl-D-aspartate receptors following anodal polarization in the rat brain.

c-Fos protein-like immunoreactivity (IR) was investigated in the rat brain following an application of weak anodal direct current to the surface of the unilateral sensorimotor cortex in an attempt to elucidate the cellular and molecular bases of central plasticity. Anodal polarization resulted in a massive increase in c-Fos protein-like IR in neurons of the cingulate, piriform, frontoparietal cortices, and hippocampus ipsilateral to the polarization. The effects were dependent upon the duration and intensity of currents applied. The time-dependent induction of c-Fos protein-like IR was maximal at 1 h, became weaker by 6 h, and almost returned to the baseline within 24 h following polarization. When MK-801 [(+)-5-methyl-10,11-di-hydro-5H- dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate], a noncompetitive antagonist for N-methyl-D-aspartate (NMDA) receptors, was injected intraperitoneally, the induction of this nuclear protein was reduced or completely blocked in both hemispheres, except around the polarized point itself, as a function of the time and dosage. These results suggest that the proto-oncogene c-fos is rapidly and transiently activated in the brain following anodal polarization and this activation is mediated by NMDA receptors.

Animals↗

Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain.

This study was designed to quantify the microglial response following an injection of N-methyl-D-aspartate (NMDA) into the sensorimotor cortex of 6-day-old rats. After survival times ranging from 10 h to 28 days, cryostat sections were processed for the demonstration of microglial cells by means of tomato lectin histochemistry. The injection of NMDA caused an extensive primary lesion involving the neocortex, the rostral hippocampus, and rostral thalamus. In addition, secondary retrograde/anterograde degeneration was also observed in the ventrobasal (VB) complex of the thalamus. Microglial reactivity was already present at 10 h postlesion and restricted to areas of neuronal degeneration. Quantitative analysis was performed on digitized images using NIH Image software and a Macintosh computer. The method is based on densitometric ratios, referred to as the "reactivity grade," between the ipsilateral lesion side and the contralateral control side. Measurements were made to determine a possible increase in the number of microglial cells as well as an increase in lectin binding. The analysis showed that microglial reactivity in areas of primary degeneration peaked at 3 days postlesion, when it was significantly (P < 0.01) higher in comparison to saline-injected litter mates. Microglial response in the cerebral neocortex, showing the highest reactivity grade, as well as in other areas of primary degeneration, returned to control levels by Day 7. Microglial response in the VB complex also peaked at Day 3 (P < 0.05) but maintained this level of reactivity until 7 days postlesion (P < 0.01).

Animals↗

Use-dependent exaggeration of brain injury: is glutamate involved?

Extreme overreliance on the impaired forelimb following unilateral lesions of the forelimb representation area of the rat sensorimotor cortex (FL-SMC) leads to exaggeration of the initial cortical injury. Glutamate has repeatedly been implicated in the secondary processes leading to neuronal death following traumatic insult, chiefly because of the neuroprotective properties of excitatory amino acid antagonists in a variety of animal models of brain injury. The present study investigated the possibility that NMDA receptor-mediated processes are involved in use-dependent exaggeration of neuronal injury. Rats were fitted with one-sleeved casts that immobilized the intact forelimb for the first 7 days following FL-SMC lesion, a procedure previously shown to result in use-dependent exaggeration of injury and more severe and persistent limb-use deficits. In the present investigation, administration of MK-801 (1 mg/kg ip once daily on alternate days) during the casting period spared neural tissue surrounding the lesion and enhanced functional recovery of the impaired forelimb. These results suggest a role for NMDA receptor-mediated processes in use-dependent exaggeration of injury.

Analysis of Variance↗

Behavioral and metabolic changes in immature rats during seizures induced by homocysteic acid: the protective effect of NMDA and non-NMDA receptor antagonists.

Bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA) (600 nmol on each side) to immature 12-day-old rats induced generalized clonic-tonic seizures, recurring frequently for at least 90 min, with a high rate of survival. Electrographic recordings from sensorimotor cortex, hippocampus, and striatum demonstrated isolated spikes in the hippocampus and/or striatum as the first sign of dl-HCA action. Generalization of epileptic activity occurred during generalized clonic-tonic seizures, but electroclinical correlation was very low; dissociation between EEG pattern and motor phenomena was common. Seizures were accompanied by large decreases of cortical glucose and glycogen and by approximately 7- to 10-fold accumulation of lactate. ATP and phosphocreatine (PCr) levels remained unchanged even during longlasting (3 h) convulsions. Metabolite levels became normalized during the recovery period (24 h). The examination of the effect of selected antagonists of NMDA [AP7 (18.5 and 37 mg/kg, respectively), MK-801 (0.5 mg/kg)] and non-NMDA [NBQX (10, 15 and 30 mg/kg, respectively)] receptors revealed that seizures could be attenuated or prevented (depending on the dose employed) by antagonists of both NMDA and non-NMDA receptors, as evaluated not only according to the suppression of behavioral manifestations of seizures, but also in terms of the protection of metabolite changes accompanying seizures. All antagonists employed, when given alone in the same doses as those used for seizure protection, did not influence metabolite levels, with the exception of increased glucose concentrations. Furthermore, the pronounced anticonvulsant effect could be achieved by the combined treatment with low subthreshold doses of NMDA (AP7) and non-NMDA (NBQX) receptor antagonists, which may be of potential significance for a new approach to the treatment of epilepsy.

2-Amino-5-phosphonovalerate↗

Global sex differences in stress-induced activation of cerebral metabolism revealed by 2-deoxyglucose autoradiography.

Although it is well known that there are sex differences in stress-induced activation of the hypothalamic-pituitary-adrenal axis, it is not known if there are also gender-related differences in stress-induced neural activity. In this study, restraint and formalin injections into a forelimb were used as stressors and 2-[14C]-deoxyglucose (2DG) autoradiography was used to evaluate regional brain glucose metabolism, an index of neural activity. Analysis of blood samples collected during the 2DG procedure confirmed that stress elevates plasma glucose levels signficantly more in females than in males. Moreover, females show higher brain glucose utilization in all regions examined, including sex hormone-responsive regions such as the medial amygdala, medial preoptic nucleus, ventromedial nucleus, and arcuate nucleus, as well as the CA1 layer and dentate region of the hippocampus, the posterior parietal (sensorimotor) cortex, medial and lateral habenula, and splenium of the corpus callosum. The sex differences are apparent regardless of whether animals were injected with saline or formalin. Interestingly, the medial preoptic area, which shows robust neuroanatomical sex differences, demonstrates greater activation in response to formalin than to saline only in females. In some regions of both males and females, glucose utilization was higher on the side of the brain contralateral to the saline or formalin injection site. These findings suggest that there are widespread, gender-related differences in neuronal as well as endocrine activation in response to highly stressful conditions.

Animals↗

Brain representation of active and passive movements.

During active and passive (driven by a torque motor) flexion and extension of the right elbow, regional cerebral blood flow (rCBF) was measured in six healthy, male volunteers using positron emission tomography and the standard H2(15)O injection technique. During active as well as during passive movements of the right elbow there were strong increases in rCBF, identical in location, amount, and extent in the contralateral sensorimotor cortex. There were activations during both conditions in the supplementary motor area (stronger and more inferior in the active condition) and inferior parietal cortex (on the convexity during active movements and in the depth of the central sulcus during passive movements). During active movements only, activations of the basal ganglia and the cingulate gyrus were found. Brain activations during motor tasks are largely related to the processing of afferent information.

Adult↗

Visuomotor transformations for reaching to memorized targets: a PET study.

Positron emission tomography (PET) was used to identify cortical and subcortical regions involved in the control of reaching to visual targets. Regional cerebral blood flow (rCBF) was measured in eight healthy subjects using H2(15)O PET during the performance of three different tasks. All tasks required central fixation while a 400-ms target was flashed every 5 s at a random location around a virtual circle centered on the fixation target. Additional instructions differed according to the task: (i) visual detection of the target without overt responses; (ii) immediate pointing to the most recent target in the sequence, and (iii) pointing to the previous target in the sequence. By design, the two motor tasks differed in the cognitive processing required. In each trial of immediate pointing, the spatial location of only the most recent target needed to be processed. In each trial of pointing to the previous, instead, while the most recent target was stored in memory for the movement of the next trial, the previous target had to be retrieved from memory to direct the current movement. Limb trajectories were comparable between the two motor tasks in terms of most spatiotemporal parameters examined. Significant rCBF increases were identified using analysis of covariance and t statistics. Compared with visual detection there was activation of primary sensorimotor cortex, ventrolateral precentral gyrus, inferior frontal gyrus in the opercular region, supramarginal gyrus, and middle occipital gyrus, all these sites in the hemisphere (left) contralateral to the moving limb, and cerebellar vermis, during both immediate pointing and pointing to the previous. During immediate pointing there was additional activation of left inferior parietal lobule close to the intraparietal sulcus, and when compared with pointing to the previous, dorsolateral prefrontal cortex bilaterally. During pointing to the previous, instead, there was additional activation of supplementary motor cortex, anterior and midcingulate, and inferior occipital gyrus in the left hemisphere; superior parietal lobule, supramarginal gyrus, and posterior hippocampus in the right hemisphere; lingual gyri and cerebellar hemispheres bilaterally; anterior thalamus; and pulvinar. The activation of two partially distinct cerebral networks in these two motor tasks reflects the different nature of signal processing involved. In particular, the specific activation of intraparietal sulcus and prefrontal cortex in immediate pointing appears characteristic of a network for visuospatial working memory. By contrast, the corticolimbic network engaged in pointing to the previous could mediate spatial attention and the sequence of encoding, recording, and decoding of spatial memories required by a dual task with two competing targets.

Adult↗

The effect of normal aging on the coupling of neural activity to the bold hemodynamic response.

The use of functional neuroimaging to test hypotheses regarding age-related changes in the neural substrates of cognitive processes relies on assumptions regarding the coupling of neural activity to neuroimaging signal. Differences in neuroimaging signal response between young and elderly subjects can be mapped directly to differences in neural response only if such coupling does not change with age. Here we examined spatial and temporal characteristics of the BOLD fMRI hemodynamic response in primary sensorimotor cortex in young and elderly subjects during the performance of a simple reaction time task. We found that 75% of elderly subjects (n = 20) exhibited a detectable voxel-wise relationship with the behavioral paradigm in this region as compared to 100% young subjects (n = 32). The median number of suprathreshold voxels in the young subjects was greater than four times that of the elderly subjects. Young subjects had a slightly greater signal:noise per voxel than the elderly subjects that was attributed to a greater level of noise per voxel in the elderly subjects. The evidence did not support the idea that the greater head motion observed in the elderly was the cause of this greater voxel-wise noise. There were no significant differences between groups in either the shape of the hemodynamic response or in its the within-group variability, although the former evidenced a near significant trend. The overall finding that some aspects of the hemodynamic coupling between neural activity and BOLD fMRI signal change with age cautions against simple interpretations of the results of imaging studies that compare young and elderly subjects.

Adolescent↗

Accuracy and limitation of functional magnetic resonance imaging for identification of the central sulcus: comparison with magnetoencephalography in patients with brain tumors.

The aim of the present study was to clarify the accuracy and limitation of functional magnetic resonance imaging (fMRI) for the identification of the central sulcus affected by brain tumors. Twelve normal volunteers and 11 patients with intracranial tumors adjacent to the central sulcus underwent fMRI and magnetoencephalography (MEG). Three patients were evaluated again after surgery. fMRI was performed with a 1.5 Tesla scanner during repetitive opening and closing of each hand. Cross-correlation function was used to identify activation areas, and the central sulcus was defined as the nearest sulcus to the highest activation spots that were determined by elevating correlation coefficient threshold. Somatosensory-evoked fields were measured using a whole head MEG system. The central sulcus was defined as the nearest sulcus to the N20m for the median nerve stimulus. fMRI and MEG coincided in defining the central sulcus in all 24 hemispheres of volunteers and all 10 examined nonaffected hemispheres of patients. The fMRI-defined central sulcus coincided with the MEG-defined central sulcus in nine (82%) but did not in two (18%) affected hemispheres of patients. The preoperative mismatch disappeared after surgery in one of the two patients. The present study indicates that fMRI successfully defined the central sulcus in most of the patients with brain tumors. However, in a few cases, fMRI was not reliable probably due to venous flow changes by tumor compression and/or compensational activity by brain tissues surrounding the primary sensorimotor cortex. For precise functional assessment of the brain affected by intracranial tumors, combination of fMRI and MEG will be recommended.

Adult↗

Regional variability of cerebral blood oxygenation response to hypercapnia.

In functional magnetic resonance imaging studies changes in blood oxygenation level-dependent (BOLD) signal intensities during task activation are related to multiple physiological parameters such as cerebral blood flow, volume, and oxidative metabolism, as well as to the regional microvascular anatomy. Consequently, the magnitude of activation-induced BOLD signal changes may vary regionally and between subjects. The aim of this study was to use a uniform global stimulus such as hypercapnia to quantitatively investigate the regional BOLD response in the human brain. In 10 healthy volunteers, T2*-weighted gradient echo images were acquired for a total dynamic scanning time of 9 min during alternating periods of breath holding for 30 s after expiration and self-paced normal breathing for 60 s. Hypercapnia-induced BOLD signal changes in the sensorimotor cortex, frontal cortex, basal ganglia, visual cortex, and cerebellum were significantly different (P < 0.001) and varied from 1.8 to 5.1%. The highest BOLD signal changes were found in the cerebellum and visual cortex, whereas the lowest BOLD signal increase was observed in the frontal cortex. These results demonstrate a regional dependence of the BOLD signal changes during breath hold-induced hypercapnia, indirectly supporting the notion of regional different sensitivities of BOLD responses to task activation.

Adult↗

The functional neuroanatomy and long-term reproducibility of brain activation associated with a simple finger tapping task in older healthy volunteers: a serial PET study.

We examined long-term reproducibility of the functional organization of the brain associated with a simple finger tapping movement using positron emission tomography (PET). Repeat measurements of regional cerebral blood flow were obtained in 10 individuals, ages 35 to 82 years (mean 52 years), at scanning sessions separated by 6 months. Although the functional neuroanatomy of hand movements has previously been investigated with PET by a number of groups, none has reported systematic investigation of the consistency of brain activation over an extended time. As expected, we found significant activation in the left precentral gyrus [Talairach coordinate (-32, -34, 52)], postcentral gyrus (-22, -48, 56), and supplementary motor area (SMA) (-2, -18, 52) at the initial study, consistent with previous studies in younger subjects. For the follow-up study we also found significant activation in the left precentral (-36, -28, 52) and postcentral (-28, -36, 52) gyri and in the SMA (2, -16, 56). Our group results demonstrate consistent anatomical location and extent of motor activation over time. More importantly, analysis of individuals confirmed the presence of consistent sites of activation in primary sensorimotor cortex and SMA over the 6-month interval in most subjects. A high degree of consistency in location of activation in the group, and within individuals, over time suggests that changes in loci of activation may be confidently monitored using the PET method. Evidence of individual differences in extent of activation over time highlights the need for caution when interpreting similar changes in patient studies.

Adult↗

Gender difference in premotor activity during active tactile discrimination.

To investigate possible gender differences in tactile discrimination tasks, we measured cerebral blood flow of seven men and seven women using positron emission tomography and (15)O water during tactile tasks performed with the right index finger. A nondiscrimination, somatosensory control task activated the left primary sensorimotor cortex and the left parietal operculum extending to the posterior insula without any gender difference. Compared with the control task, discrimination tasks activated the superior and inferior parietal lobules bilaterally, right dorsal premotor cortex, and dorsolateral prefrontal cortex in both genders, consistent with the notion of right hemisphere involvement during exploratory attentional movements. In both genders, symmetric activation of the superior and inferior parietal lobules and asymmetric activation of the right dorsolateral prefrontal cortex were confirmed. The former is consistent with the spatial representation of the tactile input and the latter with the spatial working memory. However, activation of the dorsal premotor cortex was asymmetric in men, whereas it was symmetric in women, the gender difference being statistically significant. This may suggest gender differences in motor programs for exploration in manipulospatial tasks such as tactile discrimination with active touch, possibly by greater interhemispheric interaction through the dorsal premotor cortices in women than in men.

Adult↗

Anatomic and functional variability: the effects of filter size in group fMRI data analysis.

In the analysis of group fMRI scans, an optimal spatial filter should be large enough to accurately blend functionally homologous anatomic regions, yet small enough not to blur the functionally distinct regions. Hanning filters varying from 0.0 to 18.0 mm were evaluated in a group analysis of six healthy controls performing a simple finger-tapping paradigm. Test-retest reliability and Talairach-based measurements of the sensorimotor region were used to explore the optimal filter size. Two distinct regions of functional activation were noted in the sensorimotor cortex in group images (n = 6) at both time 1 and time 2. These regions merge once the filter size exceeds approximately 6.0 mm. The original hypothesis that these represented a motor and sensory activation was rejected on the basis of structural and functional variability. A discussion of the inherent difficulties in choosing an appropriate filter size is presented.

Adult↗

Changes of cerebral blood flow, oxygenation, and oxidative metabolism during graded motor activation.

In the present studies fMRI and a hypercapnic calibration procedure were used to monitor simultaneous changes in cerebral blood flow (CBF), cerebral blood oxygenation, and cerebral metabolic rate of oxygen (CMRO(2)) during activation in the sensorimotor cortex. In the first set of experiments seven volunteers performed bilateral, self-paced finger tapping and in the second set of experiments six volunteers performed bilateral finger tapping with six different frequencies (0.5-3 Hz). During the latter task relative CBF and BOLD signal intensity changes varied linearly as a function of stimulus frequency. In good agreement with recent PET and fMRI data increases in CMRO(2) were smaller than the corresponding changes in CBF during self-paced finger tapping and at all levels of graded motor activation. At a single level of activation and during graded activation there was a positive linear relationship between CBF and CMRO(2) with ratios of approximately 3:1. Comparable proportionality constants have been found in the visual cortex and primary sensory cortex, indicating similarities between the relationship of CBF and CMRO(2) in various cortical regions.

Adult↗

Delayed onset of potentiation in neocortical EPSPS during long-term potentiation (LTP)--a postsynaptic mechanism or heterogeneous synaptic inputs?

Long-term potentiation (LTP) is an activity-dependent, long-lasting change in the efficacy of synaptic transmission, which is a possible neural substrate for learning and memory. We have induced LTP at some synapses in slices of rat sensorimotor cortex in vitro, by using either brief tetanic stimulation of afferent axons or an associative, postsynaptic method of induction. We were successful in inducing LTP of excitatory postsynaptic potentials (EPSPs) in 11 out of 53 neurones in layers III, V or VI. In view of the importance of N-methyl-D-aspartate (NMDA) receptor activation for the induction of LTP in neocortex, as in regions of hippocampus, we hypothesized that the failures of induction of LTP might be due to a lack of NMDA-receptor mediated activity in the afferent pathway onto layer V and VI neurones. Hence we bath-applied the selective NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and examined its effect on composite PSPs evoked in layer V and VI neurones. We found that 94% of PSPs (44/47) were partly mediated by NMDA-receptor activity, so a lack of functioning NMDA receptors was not likely to be the reason for failures of induction of LTP by afferent tetanic stimulation. When we induced LTP (in the absence of AP5) and applied AP5 15 to 20 min after the establishment of LTP, the expression of LTP was reversibly reduced: during LTP, synaptic responses consisted of an early AP5-insensitive component and a later AP5-sensitive component. This was seen in both EPSPs and field potentials in layers V and VI. Although the depolarising slope of most of the neocortical EPSPs was increased during the expression of LTP, we found that in 9/11 short latency EPSPs the onset of the potentiated component of the EPSP was delayed by a few ms with respect to the onset of the EPSP. In 7 of the 9 EPSPs the LTP was induced postsynaptically by pairing an evoked EPSP repetitively with intracellular depolarising pulses. The explanation for the delayed onset of the potentiated component in these EPSPs could be either that a postsynaptic voltage-sensitive mechanism was involved, or that the afferent terminals on the neurons were heterogeneous. The afferents responsible for the shortest latency components of the neocortical EPSPs may not be capable of sustaining LTP.

2-Amino-5-phosphonovalerate↗

Repeated fluorodeoxyglucose positron emission tomography of the brain in infants with suspected hypoxic-ischaemic brain injury.

Positron emission tomography (PET) permits the study of cerebral metabolism in vivo. We performed repeated PET studies with fluorine-18 fluorodeoxyglucose (FDG) as a tracer to measure cerebral glucose metabolism for estimation of neurological prognosis in infants with suspected hypoxic-ischaemic brain injury. Fourteen infants (gestational age 35.3 +/- 4.67 weeks) were examined during the neonatal period (at age 38.4 +/- 2.7 weeks) and again at the age of 3.5 +/- 0.7 months; one further infant was studied only once at the age of 2.5 months. All children also underwent ultrasound examinations. Electroencephalography and computed tomography or magnetic resonance imaging were performed according to their clinical condition and their neurological development has been followed. FDG accumulated most actively in the subcortical areas (thalami, brainstem and cerebellum) and the sensorimotor cortex during the neonatal period. The repeated PET study showed that the uptake of FDG was markedly high and increased in all brain sections of infants with normal development (n = 11), whereas those with delayed development (n = 4) had significantly lower values (P < or = 0.005).

Brain Ischemia↗

Individual corticorubral neurons project bilaterally during postnatal development and following early contralateral cortical lesions.

The corticorubral projections in adult cats are primarily uncrossed. However, early in development and after early unilateral lesions of the sensorimotor cortex, crossed corticorubral projections are also observed. The present study was performed to disclose (1) whether the crossed projections originate from neuronal subpopulations different from those producing uncrossed ones and (2) how the neurons that give rise to the crossed projections in the lesioned animals are related to those occurring in normal development. We injected fluorescent latex microspheres into the red nucleus of two groups of animals: (1) intact kittens at postnatal week 3 and (2) kittens that had received unilateral ablation of the cerebral cortex at this stage and were then allowed to survive for at least 4 weeks. Red fluorescing microspheres were injected on one side and green ones on the other. In both normal and lesioned kittens, a number of cells in the cortex were labeled as a result of the contralateral as well as the ipsilateral injections, and no difference in size or distribution was found between the cells labeled from contralateral and ipsilateral injections. More than half of the cells labeled from contralateral injections were double-labeled in both groups of animals. These results indicate that individual corticorubral cells project bilaterally in normal development as well as following unilateral lesions of the cortex. With respect to the cells producing crossed projections, they were similar in both laminar and regional distributions between the intact and lesioned animal, suggesting that the crossed projections arise from the same neuronal subpopulation before and after cortical lesions. This view was supported by sequential injections of the tracers, which indicated that cells normally projecting contralaterally maintained the crossed projection after the lesions. Taking into account our previous observations that growth and proliferation of crossed corticorubral axons took place in the red nucleus (Murakami et al. 1991a), it is likely that growth and proliferation of the axons in denervated targets play a major role in lesion-induced establishment of aberrant projections.

Aging↗

Amino acid immunoreactivity in corticospinal terminals.

Anterogradely labeled corticospinal axons and their terminals were identified after injections of wheat germ-agglutinin conjugated to horseradish peroxidase in the sensorimotor cortex of rats. Thin myelinated axons were labeled in the corticospinal tract. Terminal labeling was densest in the internal basilar nucleus and laminae III-IV of the dorsal horn throughout the spinal cord; electron microscopical observations were mainly from the cervical enlargement. Labeled terminals were most often small and dome-shaped with densely packed, clear round vesicles and sparse mitochondria. These terminals established asymmetric synapses with small dendrites or spines and were never involved in axoaxonic contacts. Postembedding immunocytochemistry was used to study the subcellular distribution of glutamate, aspartate, and gamma-aminobutyric acid (GABA). Corticospinal terminals appeared enriched in glutamate, but not GABA. Some corticospinal terminals appeared enriched in aspartate, though the labeling was less selective than in the case of glutamate. GABA immunolabeling was very dense in about 20% of terminals. These were most often small, rich in mitochondria, and made symmetric synapses; they were not anterogradely labeled from the cortex. Quantitative analysis on double immunolabeled material allowed a direct comparison of particle density for different antigens in the same section. Terminals with a high density of particles coding for glutamate were not enriched with GABA, and terminals immunolabeled for GABA were not enriched with glutamate. There was no significant correlation between glutamate and aspartate immunolabeling in corticospinal terminals; a subpopulation of these terminals may be enriched in aspartate. Aspartate immunolabeling was consistently higher in dendrites postsynaptic (in the plane of section) to corticospinal terminals than in other dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗