Parvalbumin Content Research Articles

Measuring parvalbumin levels in fish muscle tissue: Relevance of muscle locations and storage conditions

Fish is an allergenic food capable of provoking severe anaphylactic reactions. Parvalbumin is the major allergen identified in fish and frog muscles. Antibodies against fish and frog parvalbumin have been used to quantify parvalbumin levels from fish. However, these antibodies react variably with parvalbumin from different fish species. Several factors might be responsible for this variation including instability of parvalbumin in fish muscle as a result of frozen storage and differential parvalbumin expression in muscles from various locations within the whole fish. We aimed to investigate whether these factors contribute to the previously observed variable immunoreactivity of the anti-parvalbumin antibodies. Results showed the detection of parvalbumin by these antibodies was unaffected by frozen storage of muscles for 112days. However, the parvalbumin content decreased in fish muscles from anterior to posterior positions. This factor may partially explain for the inconsistent reactivity of anti-parvalbumin antibodies to different fish species.

Control of Neuronal Excitability by Calcium Binding Proteins: A New Mathematical Model for Striatal Fast-Spiking Interneurons

Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, where PV is only expressed in the fast-spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS neurons did not allow the study of the coupling between PV concentration and electrical activity. In the present paper, we propose a new mathematical model for the striatal FS interneurons that includes apamin-sensitive small conductance Ca2+-dependent K+ channels (SK) and the presence of a calcium buffer. Our results show that a variation in the concentration of PV can modulate substantially the intrinsic excitability of the FS interneurons and therefore may be involved in the information processing at the striatal level.

Comparative Analyses of Allergens between Landlocked and Anadromous Species of Masu Salmon Oncorhynchus masou masou

Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8-7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28-0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).

How the 'slow' Ca2+ buffer parvalbumin affects transmitter release in nanodomain-coupling regimes

Parvalbumin is thought to act in a manner similar to EGTA, but how a slow Ca(2+) buffer affects nanodomain-coupling regimes at GABAergic synapses is unclear. Direct measurements of parvalbumin concentration and paired recordings in rodent hippocampus and cerebellum revealed that parvalbumin affects synaptic dynamics only when expressed at high levels. Modeling suggests that, in high concentrations, parvalbumin may exert BAPTA-like effects, modulating nanodomain coupling via competition with local saturation of endogenous fixed buffers.

Tail muscle parvalbumin content is decreased in chronic sacral spinal cord injured rats with spasticity

In rats, chronic sacral spinal isolation eliminates both descending and afferent inputs to motoneurons supplying the segmental tail muscles, eliminating daily tail muscle EMG activity. In contrast, chronic sacral spinal cord transection preserves afferent inputs, causing tail muscle spasticity that generates quantitatively normal daily EMG. Compared with normal rats, rats with spinal isolation and transection/spasticity provide a chronic model of progressive neuromuscular injury. Using normal, spinal isolated and spastic rats, we characterized the activity dependence of calcium-handling protein expression for parvalbumin, fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) and slow SERCA2. As these proteins may influence fatigue resistance, we also assayed the activities of oxidative (citrate synthase; CS) and glycolytic enzymes (glyceraldehyde phosphate dehydrogenase; GAPDH). We hypothesized that, compared with normal rats, chronic isolation would cause decreased parvalbumin, SERCA1 and SERCA2 expression and CS and GAPDH activities. We further hypothesized that chronic spasticity would promote recovery of parvalbumin, SERCA1 and SERCA2 expression and of CS and GAPDH activities. Parvalbumin, SERCA1 and SERCA2 were quantified with Western blotting. Citrate synthase and GAPDH activities were quantified photometrically. Compared with normal rats, spinal isolation caused large decreases in parvalbumin (95%), SERCA1 (70%) and SERCA2 (68%). Compared with spinal isolation, spasticity promoted parvalbumin recovery (ninefold increase) and a SERCA2-to-SERCA1 transformation (84% increase in the ratio of SERCA1 to SERCA2). Compared with normal values, CS and GAPDH activities decreased in isolated and spastic muscles. In conclusion, with complete paralysis due to spinal isolation, parvalbumin expression is nearly eliminated, but with muscle spasticity after spinal cord transection, parvalbumin expression partly recovers. Additionally, spasticity after transection causes a slow-to-fast SERCA isoform transformation that may be compensatory for decreased parvalbumin content.

Glutamate and nicotinic receptor interactions in working memory: importance for the cognitive impairment of schizophrenia

This article reaches across disciplines to correlate results in molecular, cellular, behavioral, and clinical research to develop a more complete picture of how working memory (WM) functions. It identifies a new idea that deserves further investigation. NMDA glutamate receptors (NMDAR) are critical for memory function. NMDAR inhibition effectively reproduces principal manifestations of schizophrenia (SP), such as WM impairment and GABAergic deficit (mainly reduction of glutamic acid decarboxylase 67 (GAD67) and parvalbumin (PV) content). Nicotine and selective α7 nicotinic acetylcholine receptor (nAChR) agonists reduce WM impairments in patients with SP and reverse WM deficits in animals treated with NMDAR antagonists. The mechanism of this effect is unknown. Importantly, WM recovery occurs even before restoration of NMDAR blockade-induced molecular alterations, including reduced GAD67 in interneurons. Our insight into the cognitive-enhancing effect of α7 nAChR agonists, particularly in the animal models of SP, combines reviews of recent findings on glutamate and nicotinic receptor expression in the neuronal circuits involved in WM, the properties of these receptors, their implication in WM regulation, generation of rhythmic neuronal activity, resulting in a proposed hypothesis for further investigations. We suggest that (1) cortical/hippocampal interneurons, particularly PV positive, play a crucial role in WM and that impairment of these cells in SP could be behind the WM deficit; (2) activation of α7 nAChRs could restore calcium signaling and intrinsic properties of these interneurons, and associated with these events, computational capacity, gamma rhythmic activity, and WM would also be restored.

A21 Altered calcium kinetics in skeletal muscle fibres of the R6/2 mouse model of HD

Background and aimsSome of the most obvious peripheral tissue changes in Huntington's disease (HD) are found in skeletal muscle. Alterations in morphology, gene expression pattern, energy metabolism and differentiation have...

Important Variations in Parvalbumin Content in Common Fish Species: A Factor Possibly Contributing to Variable Allergenicity

Background: Although 95% of fish-allergic patients are sensitized to the major fish allergen parvalbumin, clinical reactions to different fish species vary considerably in symptoms, intensity and frequency in allergic subjects. This study aimed at the quantification of parvalbumin levels in salmon, trout, cod, carp, mackerel, herring, redfish and tuna. Methods: Fish muscle extracts were separated by SDS-PAGE and parvalbumin content was estimated by densitometric band quantification. Individual antisera were raised in BALB/c mice against parvalbumins purified from seven fish species. Parvalbumin content was quantified in fish (raw/processed) and skin prick test (SPT) solutions by ELISA using the corresponding anti-serum for detection and the purified parvalbumins as standards. Results: Using SDS-PAGE scanning, parvalbumin contents were <0.5 mg per gram tissue for mackerel, 0.5–2 mg for salmon and trout, and >2 mg for cod, carp, redfish and herring. Using ELISA, parvalbumin content ranged from <0.05 mg for tuna, 0.3–0.7 mg for mackerel, 1–2.5 mg for salmon, trout and cod to >2.5 mg per gram raw muscle for carp, herring and redfish. The parvalbumin content of processed samples (cooked/commercial) was 20–60% lower. Allergen content in SPT samples ranged from 20 to 70 µg parvalbumin/ml of extract. No parvalbumin was found in tuna SPT solution. Conclusion: The parvalbumin content of most commonly consumed fish species varies considerably. Differences range from severalfold to one hundredfold. This has to be taken into account when designing food challenge tests and advising fish-allergic patients.

Parvalbumin-positive GABAergic interneurons are increased in the dorsal hippocampus of the dystrophic mdx mouse

Duchenne muscular dystrophy (DMD) is characterized by variable alterations of the dystrophin gene and by muscle weakness and cognitive impairment. We postulated an association between cognitive impairment and architectural changes of the hippocampal GABAergic system. We investigated a major subpopulation of GABAergic neurons, the parvalbumin-immunopositive (PV-I) cells, in the dorsal hippocampus of the mdx mouse, an acknowledged model of DMD. PV-I neurons were quantified and their distribution was compared in CA1, CA2, CA3, and dentate gyrus in wild-type and mdx mice. The cell morphology and topography of PV-I neurons were maintained. Conversely, the number of PV-I neurons was significantly increased in the mdx mouse. The percent increase of PV-I neurons was from 45% for CA2, up to 125% for the dentate gyrus. In addition, the increased parvalbumin content in the mdx hippocampus was confirmed by Western blot. A change in the hippocampus processing abilities is the expected functional counterpart of the modification displayed by PV-I GABAergic neurons. Altered hippocampal functionality can be responsible for part of the cognitive impairment in DMD.

No evidence for enhanced parvalbumin concentration in light muscle of transgenic coho salmon (Oncorhynchus kisutch)

The expression of parvalbumin, a major allergenic protein of fish muscle, was determined in rapidly growing transgenic coho salmon (Oncorhynchus mykiss) containing the growth hormone (GH) gene construct OnMTGH1. Three different methods for parvalbumin analysis were used: (1) measurement of the mRNA concentration by real-time RT-PCR; (2) isoelectric focusing of sarcoplasmic proteins; (3) assessment of amount of heat-stable, Ca2+-binding sarcoplasmic proteins by measuring absorbance spectra of “stains-all”–protein complexes. Compared to non-transgenic coho salmon, no indication for enhanced expression of parvalbumin in transgenic fish was found either at the mRNA or at the protein level.

Is high concentration of parvalbumin a requirement for superfast relaxation?

It is generally thought that the rapid relaxation of fast muscles is facilitated by the Ca(2+) binding protein parvalbumin (Parv). Indeed superfast swimbladder (SWB) muscle of toadfish contains the largest concentration of this protein ever observed (up to 1.5 mM). At 15 degrees C toadfish perform a 100 Hz call, 400 ms in duration, followed by a long (5-15 s) intercall interval. It has been proposed that Parv helps sequester the Ca(2+) during the call, and then Ca(2+) unbinds and is pumped back into the sarcoplasmic reticulum during the long intercall interval. Midshipman (Porichthys notatus) is another fish which calls at a high frequency; 80-100 Hz at a temperature of 12-15 degrees C. However, unlike toadfish, midshipman call with a 100% duty cycle. Without an intercall interval, Parv would seem of little use as it would become saturated early in calling. Here we show that the midshipman SWB has only about 1/8th of the Parv in toadfish. Moreover, total Parv content in calling male midshipman SWB was not different from that in the non-calling female and the much slower locomotory muscles. These data suggest that Parv does not play a large role in the calling of midshipman, which is accomplished without a high concentration of this protein. Native gel-electrophoresis also revealed presence of three major (PA-I, PA-II and PA-III) and two minor (PA-Ia and PA-IIIa, <5% of total content) Parv isoforms in adult toadfish SWB. Midshipman SWB contained about equal amounts of PA-I and PA-II and also a small (approximately 10%) amount of PA-III. By amino acid composition, toadfish PA-Ia and PA-I isoforms were different from PA-II and PA-III isoforms (by 24 and 14 residues, respectively).

Changes of parvalbumin expression in the spinal cord after peripheral inflammation

Parvalbumin (PV) is a calcium-binding protein that is expressed by numerous neuronal subpopulations in the central nervous system. Staining for PV was often used in neuroanatomical studies in the past. Recently, several studies have suggested that PV acts in neurons as a mobile endogenous calcium buffer that affects temporo-spatial characteristics of calcium transients and is involved in modulation of synaptic transmission. In our experiments, expression of PV in the lumbar dorsal horn spinal cord was evaluated using densitometric analysis of immunohistological sections and Western-blot techniques in control and arthritic rats. There was a significant reduction of PV immunoreactivity in the superficial dorsal horn region ipsilateral to the arthritis after induction of the peripheral inflammation. The ipsilateral area and intensity of PV staining in this area were reduced to 38 % and 37 %, respectively, out of the total PV staining on both sides. It is suggested that this reduction may reflect decreased expression of PV in GABAergic inhibitory neurons. Reduction of PV concentration in the presynaptic GABAergic terminals could lead to potentiation of inhibitory transmission in the spinal cord. Our results suggest that changes in expression of calcium-binding proteins in spinal cord dorsal horn neurons may modulate nociceptive transmission.

Comparison of the myoplasmic calcium transient elicited by an action potential in intact fibres of mdx and normal mice

The myoplasmic free [Ca2+] transient elicited by an action potential (Delta[Ca2+]) was compared in fast-twitch fibres of mdx (dystrophin null) and normal mice. Methods were used that maximized the likelihood that any detected differences apply in vivo. Small bundles of fibres were manually dissected from extensor digitorum longus muscles of 7- to 14-week-old mice. One fibre within a bundle was microinjected with furaptra, a low-affinity rapidly responding fluorescent calcium indicator. A fibre was accepted for study if it gave a stable, all-or-nothing fluorescence response to an external shock. In 18 normal fibres, the peak amplitude and the full-duration at half-maximum (FDHM) of Delta[Ca2+] were 18.4 +/- 0.5 microm and 4.9 +/- 0.2 ms, respectively (mean +/- s.e.m.; 16 degrees C). In 13 mdx fibres, the corresponding values were 14.5 +/- 0.6 microm and 4.7 +/- 0.2 ms. The difference in amplitude is statistically highly significant (P = 0.0001; two-tailed t test), whereas the difference in FDHM is not (P = 0.3). A multi-compartment computer model was used to estimate the amplitude and time course of the sarcoplasmic reticulum (SR) calcium release flux underlying Delta[Ca2+]. Estimates were made based on several differing assumptions: (i) that the resting myoplasmic free Ca2+ concentration ([Ca2+]R) and the total concentration of parvalbumin ([Parv(T)]) are the same in mdx and normal fibres, (ii) that [Ca2+](R) is larger in mdx fibres, (iii) that [Parv(T)] is smaller in mdx fibres, and (iv) that [Ca2+]R is larger and [Parv(T)] is smaller in mdx fibres. According to the simulations, the 21% smaller amplitude of Delta[Ca2+] in mdx fibres in combination with the unchanged FDHM of Delta[Ca2+] is consistent with mdx fibres having a approximately 25% smaller flux amplitude, a 6-23% larger FDHM of the flux, and a 9-20% smaller total amount of released Ca2+ than normal fibres. The changes in flux are probably due to a change in the gating of the SR Ca2+-release channels and/or in their single channel flux. The link between these changes and the absence of dystrophin remains to be elucidated.

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.

Simulation of Ca2+ Movements within the Sarcomere of Fast-Twitch Mouse Fibers Stimulated by Action Potentials

Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca2+ spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca2+ evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca2+ complexation reactions with ATP, troponin, parvalbumin, and the SR Ca2+ pump, as well as Ca2+ transport by the pump. Results are compared with spatially averaged Ca2+ transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca2+ indicator. The furaptra ΔfCaD signal (change in the fraction of the indicator in the Ca2+-bound form) evoked by one AP is well simulated under the assumption that SR Ca2+ release has a peak of 200–225 μM/ms and a FDHM of ∼1.6 ms (16°C). ΔfCaD elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2–5, Ca2+ release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca2+ inactivation of Ca2+ release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s−1. An obvious feature of ΔfCaD during a five-shock train is a progressive decline in the rate of decay from the individual peaks of ΔfCaD. According to the simulations, this decline is due to a reduction in available Ca2+ binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca2+], the parvalbumin concentration, and possible uptake of Ca2+ by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca2+ sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.

Parvalbumin expression in trout swimming muscle correlates with relaxation rate

Rainbow trout (Oncorhynchus mykiss) display longitudinal and developmental shifts in muscle relaxation rate. This study aimed to determine the role of variations in parvalbumin content in modulating muscle relaxation. Parvalbumin is a low molecular weight protein that buffers myoplasmic Ca2+ and enhances muscle relaxation. In some fish, longitudinal variations in muscle relaxation have been linked to variations in the total amount of parvalbumin present in muscle and in the relative expression of two parvalbumin isoforms. We have demonstrated previously that anterior slow-twitch or red myotomal muscle relaxes more rapidly than that from the posterior for both rainbow and brook trout. Further, younger rainbow trout parr have faster red muscle relaxation rates than older smolts. Here we report similar results for fast-twitch or white muscle. We quantified the parvalbumin expression in red and white muscle from different body positions of rainbow trout parr and smolts and for brook trout (Salvelinus fontinalis) adults. There was a significant shift in total parvalbumin content of muscle: the faster muscle from the anterior myotome contained greater amounts of parvalbumin. For brook trout, longitudinal variation in relaxation rate was also associated with shifts in the relative expression of the two parvalbumin isoforms. The faster muscle of parr contained more parvalbumin. Lastly, trout white muscle tended to have higher levels of parvalbumin and greater levels of the Parv2 (relative to Parv1) isoform as compared to red muscle. Parvalbumin expression correlated with muscle relaxation rate in trout, although there were species-specific differences in the importance of altering total parvalbumin content versus shifts in relative parvalbumin isoform expression.

Adaptive responses to chronic creatine loading (CL) and voluntary running (Run) in rat skeletal muscle

We investigated whether CL and Run alters myosin heavy chain (MHC) based fibre types, the proteins that regulate intracellular calcium and the metabolic profile in the rat plantaris. Forty Sprague-Dawley male rats were assigned to one of four groups: CL+Sedentary (Cre-Sed); CL+Run (Cre-Run); control+Sed (Con-Sed); Con+Run (Con-Run). Cre-Run resulted in a 10% increase in type IIB fibres and a corresponding 11% decrease in type IIA fibres compared to Con-Run (P<0.03). Parvalbumin content was decreased by 75% in Cre-Sed and Cre-Run (P<0.04). No differences were observed in the fast Ca2+ ATPase isoform, SERCA1, in any of the groups (P>0.49). SERCA2 content, a slow Ca2+ ATPase isoform, was 21% and 19% lower in Cre-Sed and Cre-Run compared to Con-Sed and Con-Run, respectively (P≤0.05). Run increased citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities (P≤0.05), while phosphofructokinase and glyceraldehyde phosphate dehydrogenase activities did not change; Cr did not alter any of these enzyme contents (P>0.28). We conclude that CL during Run was able to maintain a faster fibre type while not changing the metabolic profile indicating a disconnect between coordinated fibre type conversions and metabolic phenotypic profiles. We suggest that CL alleviates the need for parvalbumin and SERCA2 expression probably due to enhanced high energy phosphate shuttling that better supports SERCA1. NSERC & AHFMR.

Comparison of allergenicity and allergens between fish white and dark muscles

Fish is one of the most frequent causes of immunoglobulin E (IgE)-mediated food allergy. Although the fish dark muscle is often ingested with the white muscle, no information about its allergenicity and allergens is available. Heated extracts were prepared from both white and dark muscles of five species of fish and examined for reactivity with IgE in fish-allergic patients by enzyme-linked immunosorbent assay (ELISA) and for allergens by immunoblotting. Cloning of cDNAs encoding parvalbumins was performed by rapid amplification cDNA ends. Parvalbumin contents in both white and dark muscles were determined by ELISA using antiserum against mackerel parvalbumin. Patient sera were less reactive to the heated extract from the dark muscle than to that from the white muscle. A prominent IgE-reactive protein of 12 kDa, which was detected in both white and dark muscles, was identified as parvalbumin. Molecular cloning experiments revealed that the same parvalbumin molecule is contained in both white and dark muscles of either horse mackerel or Pacific mackerel. Parvalbumin contents were four to eight times lower in the dark muscle than in the white muscle. The fish dark muscle is less allergenic than the white muscle, because the same allergen molecule (parvalbumin) is contained at much lower levels in the dark muscle than in the white muscle. Thus, the dark muscle is less implicated in fish allergy than the white muscle.

Parvalbumin correlates with relaxation rate in the swimming muscle of sheepshead and kingfish

Parvalbumin is a muscle protein that aids in relaxation from contraction. Parvalbumin binds myoplasmic Ca(2+) during contractions, reducing calcium concentration and enhancing relaxation. Different isoforms of parvalbumin have varying affinities for calcium, and relaxation rates in skeletal muscle may be affected by variations in the isoforms of parvalbumin expressed. This study examines the effect of expression levels of parvalbumin isoforms on relaxation rate in the sheepshead, Archosargus probatocephalus (Pisces, F. Sparidae). We measured relaxation rate of each of the three fiber types, white (fast-twitch), red (slow-twitch) and pink (intermediate), from three longitudinal body positions. Sheepshead show a significant longitudinal shift in relaxation rate in red muscle, with anterior muscle displaying faster rates of relaxation than posterior, but this pattern was not significant in the pink and white muscle. We hypothesized that patterns of parvalbumin expression determine relaxation rate along the length of the fish. The prediction is that total parvalbumin content and the relative expression of parvalbumin isoforms will differ between the anterior and posterior red muscle, but little longitudinal variation will be observed in parvalbumin expression in white and pink muscle. We successfully employed protein electrophoresis (SDS-PAGE) with western blots to identify two parvalbumin isoforms in each muscle fiber type. SDS-PAGE and densitometry were used to determine the relative expression levels of the two parvalbumin isoforms and total parvalbumin expression. Red muscle displays a significant shift, from anterior to posterior, in the relative expression of the two isoforms, both in their relative contribution and in total parvalbumin content, but white and pink muscle did not. The red muscle of southern kingfish, Menticirrhus americanus (Pisces, F. Scianidae) showed a pattern similar to the red muscle of sheepshead.

Selective GABAergic Control of Higher-Order Thalamic Relays

GABAergic signaling is central to the function of the thalamus and has been traditionally attributed primarily to the nucleus reticularis thalami (nRT). Here we present a GABAergic pathway, distinct from the nRT, that exerts a powerful inhibitory effect selectively in higher-order thalamic relays of the rat. Axons originating in the anterior pretectal nucleus (APT) innervated the proximal dendrites of relay cells via large GABAergic terminals with multiple release sites. Stimulation of the APT in an in vitro slice preparation revealed a GABA(A) receptor-mediated, monosynaptic IPSC in relay cells. Activation of presumed single APT fibers induced rebound burst firing in relay cells. Different APT neurons recorded in vivo displayed fast bursting, tonic, or rhythmic firing. Our data suggest that selective extrareticular GABAergic control of relay cell activity will result in effective, state-dependent gating of thalamocortical information transfer in higher-order but not in first-order relays.