A novel giant non-cholinergic striatal interneuron restricted to the ventrolateral striatum coexpresses Kv3.3 potassium channel, parvalbumin, and the vesicular GABA transporter.

The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.

As Verified with the Aid of Biotinylated Spermine, the Brain Cannot Take up Polyamines from the Bloodstream Leaving It Solely Dependent on Local Biosynthesis.

The importance of polyamines (PAs) for the central nervous system (CNS) is well known. Less clear, however, is where PAs in the brain are derived from. Principally, there are three possibilities: (i) intake by nutrition, release into the bloodstream, and subsequent uptake from CNS capillaries, (ii) production by parenchymatous organs, such as the liver, and again uptake from CNS capillaries, and (iii) uptake of precursors, such as arginine, from the blood and subsequent local biosynthesis of PAs within the CNS. The present investigation aimed to unequivocally answer the question of whether PAs, especially the higher ones like spermidine (SPD) and spermine (SPM), can or cannot be taken up into the brain from the bloodstream. For this purpose, a biotin-labelled analogue of spermine (B-X-SPM) was synthesized, characterized, and used to visualize its uptake into brain cells following application to acute brain slices, to the intraventricular space, or to the bloodstream. In acute brain slices there is strong uptake of B-X-SPM into protoplasmic and none in fibrous-type astrocytes. It is also taken up by neurons but to a lesser degree. Under in vivo conditions, astrocyte uptake of B-X-SPM from the brain interstitial fluid is also intense after intraventricular application. In contrast, following intracardial injection, there is no uptake from the bloodstream, indicating that the brain is completely dependent on the local synthesis of polyamines.

Middle trapezius transfer for treatment of irreparable supraspinatus tendon tears- anatomical feasibility study.

PURPOSE: The purpose of this study was to investigate the anatomical feasibility of a middle trapezius transfer below the acromion for treatment of irreparable supraspinatus tendon tears. METHODS: This study involved 20 human cadaveric shoulders in 10 full-body specimens. One shoulder in each specimen was dissected and assessed for muscle and tendon extent, force vectors, and distance to the neurovascular structures. The opposite shoulder was used to evaluate the surgical feasibility of the middle trapezius transfer via limited skin incisions along with an assessment of range of motion and risk of neurovascular injury following transfer. RESULTS: The harvested acromial insertion of the middle trapezius tendon showed an average muscle length of 11.7 ± 3.0 cm, tendon length of 2.7 ± 0.9 cm, footprint length of 4.3 ± 0.7 cm and footprint width of 1.4 ± 0.5 cm. The average angle between the non-transferred middle trapezius transfer and the supraspinatus was 33 ± 10° in the transversal plane and 34 ± 14° in the coronal plane. The mean distance from the acromion to the neurovascular bundle was 6.3 ± 1.3 cm (minimum: 4.0 cm). During surgical simulation there was sufficient excursion of the MTT without limitation of range of motion in a retracted scapular position but not in a protracted position. No injuries to the neurovascular structures were noted. CONCLUSION: Transfer of the acromial portion of the middle trapezius for replacement of an irreparable supraspinatus seems to be feasible in terms of size, vector, excursion, mobility and safety. However, some concern regarding sufficiency of transfer excursion remains as scapula protraction can increase the pathway length of the transfer. LEVEL OF EVIDENCE: Basic Science Study/Anatomical Study.

Impaired function of GABA(B) receptors in tissues from pharmacoresistant epilepsy patients.

PURPOSE: Effects of pre- and postsynaptic γ-aminobutyric acid B (GABA(B)) receptor activation were characterized in human tissue from epilepsy surgery. METHODS: Slices of human cortical tissue were investigated in a submerged-type chamber with intracellular recordings in layers II/III. Parallel experiments were performed in rat neocortical slices with identical methods. Synaptic responses were elicited with single or paired stimulations of incrementing intervals. RESULTS: Neurons in human epileptogenic tissue exhibited usually small inhibitory postsynaptic potentials (IPSP) mediated by GABA(B) receptor, verified by the sensitivity to the selective antagonist CGP 55845A. The IPSP(B) conductance averaged 5.8 nS in neurons from epileptogenic tissues and 15.9 nS in neurons from nonepileptogenic tissues (p < 0.0001). Application of baclofen caused small conductance increases in human neurons, which were linearly related to IPSP(B) conductances. Paired-pulse stimulation revealed constant synaptic responses in human temporal lobe epilepsy (TLE) slices at all interstimulus intervals (ISIs). Pharmacologically isolated IPSP(A) in the human tissue exhibited a small paired-pulse depression (average 10% at 500 ms ISI). Bicuculline-induced paroxysmal depolarization shifts (PDSs) were transiently depressed by 24% in human TLE tissue; and by 74% in rat neocortical slices (200 ms ISI; p = 0.015). The depressions of bicuculline-induced PDSs were antagonized by CGP 55845A in both species. Staining for GABA(B) receptors revealed significantly smaller numbers of immunopositive dots in human epileptogenic neurons versus human control neurons. DISCUSSION: The small IPSP(B), baclofen-conductances, and paired-pulse depression of PDSs and IPSPs in human TLE tissue indicate a reduced density of post- and presynaptic GABA(B) receptors. The reduced efficacy of presynaptic GABA(B) receptors facilitates the occurrence of repetitive synaptic activity.

Manipulating Aggregation Behavior of the Uncharged Peptide Carbetocin.

Peptides are usually administered through subcutaneous injection. For low potency drugs, this may require high concentration formulations increasing the risk of peptide aggregation, especially for compounds without any intrinsic chargeable groups. Carbetocin was used as a model to study the behavior of uncharged peptides at high concentrations. Manipulation of the aggregation behavior of 70 mg/mL carbetocin was attempted by selecting excipients which interact with hydrophobic groups in carbetocin, and cover hydrophobic surfaces and interfaces. Peptide aggregation was induced by shaking stress and followed over time. Carbetocin solutions showed significant visible particle formation already after 4 h of shaking stress. This particle formation was not due to supersaturation or phase separation but suggested a nucleated aggregation process. None of the excipients prevented carbetocin aggregation, though altered aggregation behavior was observed, such as induction of fibril formation for most, but not all, charged excipients. Sodium dodecyl sulfate was found to accelerate peptide aggregation both below and above the critical micelle concentration in half-filled vials. However, in the absence of an air headspace, sodium dodecyl sulfate above the critical micelle concentration was capable of preventing shaking-induced carbetocin aggregation. Our study highlights the complexity in rational excipient selection to stabilize uncharged peptides at high concentration.

Agmatine modulates spontaneous activity in neurons of the rat medial habenular complex-a relevant mechanism in the pathophysiology and treatment of depression?

The dorsal diencephalic conduction system connects limbic forebrain structures to monaminergic mesencephalic nuclei via a distinct relay station, the habenular complexes. Both habenular nuclei, the lateral as well as the medial nucleus, are considered to play a prominent role in mental disorders like major depression. Herein, we investigate the effect of the polyamine agmatine on the electrical activity of neurons within the medial habenula in rat. We present evidence that agmatine strongly decreases spontaneous action potential firing of medial habenular neurons by activating I1-type imidazoline receptors. Additionally, we compare the expression patterns of agmatinase, an enzyme capable of inactivating agmatine, in rat and human habenula. In the medial habenula of both species, agmatinase is similarly distributed and observed in neurons and, in particular, in distinct neuropil areas. The putative relevance of these findings in the context of depression is discussed. It is concluded that increased activity of the agmatinergic system in the medial habenula may strengthen midbrain dopaminergic activity. Consequently, the habenular-interpeduncular axis may be dysregulated in patients with major depression.

Electrophysiological properties of neurons and synapses in the lateral habenular complex (LHb).

Animal including human behavior is highly sophisticated. Besides reflective actions it is largely based on the desire for magnificent internal feelings, which are provided by the reward system. Its counterpart an "anti-reward" system is mainly composed of the lateral habenular complex (LHb) and its extensive interconnections with the monoaminergic cell groups in the mid- and hindbrain. The present review focuses on the neuronal composition and the internal signaling in the LHb. Morphologically six distinct types of neurons (spherical, fusiform-1, fusiform-2, polymorphic, vertical, neurogliaform) can be identified. In contrast, setting aside neurogliaform cells, only three broad categories (silent, tonic firing, bursting) can be identified using electrophysiological criteria. Functionally, LHb neurons express HCN channels and therefore in an "indifferent" situation LHb appears to be tonically active. When the situation takes a turn for the better habenular cells become inhibited, releasing dopaminergic VTA neurons from continuous damping. In contrast, when the situation takes a turn for the worse, LHb neurons are stimulated, completely shutting down the activity of dopaminergic cells in the VTA.

Polyanion based controlled release system for the GnRH-receptor antagonist degarelix.

The aim of this study was to investigate the interaction between the positively charged gonadotropin releasing hormone receptor antagonist degarelix and the two polyanions alginate and carboxymethyl cellulose (CMC). Light as well as transmission electron microscopy revealed that complexes formed by simple mixing of the peptide with one of the polymers had a nano-structure consisting of twisted fibers. The remarkable unique process of complex formation could be followed by isothermal titration calorimetry: We found that peptide self-aggregates dissolved upon the addition of polyanion and peptide-polymer-complexes formed thereafter with the anionic polymer as a template. Peptide release from the complexes was tested in vitro and in vivo and compared to the dissolution of drug from self-aggregates. In vitro the release was monitored over a period of three months. We could find only slight differences in the release kinetics for the alginate and the CMC complexes compared to the pure drug. An in vivo study in Sprague Dawley rats showed similar degarelix plasma concentration levels for the complex formulations and an aqueous degarelix solution following subcutaneous injection. Overall, our findings suggest a competition between complex formation and peptide aggregation, which did not increase the availability of free drug.

In vivo biocompatibility, sustained-release and stability of triptorelin formulations based on a liquid, degradable polymer.

Hexylsubstituted poly(lactic acid) (hexPLA) is a viscous polymer, which degrades in the presence of water similar to the structure related poly(lactic acid). With hydrophilic active compounds, like Triptorelin acetate, the lipophilic polymer was formulated in form of parenterally injectable suspensions. This first in vivo study toward the biocompatibility of hexPLA implants in rats over 3 months in comparison to in situ forming poly(lactic-co-glycolic acid) (PLGA) formulations is presented here. The hexPLA implants showed only a mild acute inflammation at the injection site after application, which continuously regressed. In contrast to the PLGA formulations, hexPLA did not provoke an encapsulation of the implant with extracellular matrix. Prior to the formulation application, the stability of Triptorelin inside the hexPLA matrix was assessed under different storage conditions and in the presence of buffer to simulate a peptide degrading environment. At 5°C Triptorelin showed a stability of 98% inside the polymer for at least 6 months. The stability was still 78% at an elevated temperature of 40°C. HexPLA protected the incorporated peptide from the surrounding aqueous environment, which resulted in 20% less degradation inside the polymer compared to the solution. This protection effect supports the use of Triptorelin-hexPLA formulations for parenteral sustained-release formulations. In a second in vivo evaluation in Wistar Hannover rats, formulations containing 5% and 10% Triptorelin in the polymeric matrix released the active compound continuously for 6 months. The formulations showed a higher release during the initial 7 days, which is necessary for the clinical use to down-regulate all GnRH-receptors. Afterwards, a zero order drug release was observed over the first 3 months. After 3 months, the plasma levels decreased slowly but remained at effective concentrations for the total of 6 months. Furthermore, a qualitative in vitro-in vivo correlation was observed, possibly facilitating future optimization of the Triptorelin-hexPLA sustained-release formulations.

Single processing step toward injectable sustained-release formulations of Triptorelin based on a novel degradable semi-solid polymer.

Poly(lactic acid) is a widely used polymer for parenteral sustained-release formulations. But its solid state at room-temperature complicates the formulation process, and elaborate formulation systems like microparticles and self-precipitating implants are required for administration. In contrast, hexylsubstituted poly(lactic acid) (hexPLA) is a viscous, biodegradable liquid, which can simply be mixed with the active compound. In this study, the feasibility to prepare injectable suspension formulations with peptides was addressed on the example of the GnRH-agonist Triptorelin. Two formulation procedures, of which one was a straight forward one-step cryo-milling-mixing process, were compared regarding the particle size of the peptide in the polymer matrix, distribution, and drug release. This beneficial method resulted in a homogeneous formulation with an average particle diameter of the incorporated Triptorelin of only 4.1 µm. The rheological behavior of the Triptorelin-hexPLA formulations was assessed and showed thixotropic and shear-thinning behavior. Viscosity and injectability were highly dependent on the drug loading, polymer molecular weight, and temperature. Nine formulations with drug loadings from 2.5% to 10% and hexPLA molecular weights between 1500 and 5000 g/mol were investigated in release experiments, and all displayed a long-term release for over 3 months. Formulations with hexPLA of 1500 g/mol showed a viscosity-dependent release and hexPLA-Triptorelin formulations of over 2500 g/mol a molecular weight-dependent release profile. In consequence, the burst release and rate of release were controllable by adapting the drug loading and the molecular weight of the hexPLA. The degradation characteristics of the hexPLA polymer during the in vitro release experiment were studied by following the molecular weight decrease and weight loss. Triptorelin-hexPLA formulations had interesting sustained-release characteristics justifying further investigations in the drug-polymer interactions and the in vivo behavior.

Localization of a FMRFamide-related peptide in efferent neurons and analysis of neuromuscular effects of DRNFLRFamide (DF2) in the crustacean Idotea emarginata.

In the ventral nerve cord of the isopod Idotea emarginata, FMRFamide-immunoreactive efferent neurons are confined to pereion ganglion 5 where a single pair of these neurons was identified. Each neuron projects an axon into the ipsilateral ventral and dorsal lateral nerves, which run through the entire animal. The immunoreactive axons form numerous varicosities on the ventral flexor and dorsal extensor muscle fibres, and in the pericardial organs. To analyse the neuromuscular effects of a FMRFamide, we used the DRNFLRFamide (DF2). DF2 acted both pre- and postsynaptically. On the presynaptic side, DF2 increased transmitter release from neuromuscular endings. Postsynaptically, DF2 depolarized muscle fibres by approximately 10 mV. This effect was not observed in leg muscles of a crab. The depolarization required Ca2+, was blocked by substituting Ca2+ with Co2+, but not affected by nifedipine or amiloride. In Idotea, DF2 also potentiated evoked extensor muscle contractions. The amplitude of high K+ contractures was increased in a dose dependent manner with an EC50 value of 40 nm. In current-clamped fibres, DF2 strongly potentiated contractions evoked by current pulses exceeding excitation-contraction threshold. In voltage-clamped fibres, the inward current through l-type Ca2+ channels was increased by the peptide. The observed physiological effects together with the localization of FMRFamide-immunoreactive efferent neurons suggest a role for this type of peptidergic modulation for the neuromuscular performance in Idotea. The pre- and postsynaptic effects of DF2 act synergistically and, in vivo, all should increase the efficacy of motor input to muscles resulting in potentiation of contractions.

Dopamine depresses cholinergic oscillatory network activity in rat hippocampus.

The dopaminergic neuronal system is implicated in cognitive processes in a variety of brain regions including the mesolimbic system. We have investigated whether dopamine also affects synchronized network activity in the hippocampus, which has been ascribed to play a pivotal role in memory formation. Gamma frequency (20-80 Hz) oscillations were induced by the cholinergic agonist carbachol. Oscillatory activity was examined in area CA3 of Wistar rat hippocampal slices, employing field potential and intracellular recordings. Application of carbachol initiated synchronized population activity in the gamma band at 40 Hz. Induced gamma activity persisted over hours and required GABAA receptors. Dopamine reversibly decreased the integrated gamma band power of the carbachol rhythm by 62%, while its frequency was not changed. By contrast, individual pyramidal cells recorded during carbachol-induced field gamma activity exhibited theta frequency (5-15 Hz) membrane potential oscillations that were not altered by dopamine. The dopamine effect on the field gamma activity was mimicked by the D1 receptor agonist SKF-383393 and partially antagonized by the D1 antagonist SCH-23390. Conversely, the D2 receptor agonist quinpirole failed to depress the oscillations, and the D2 antagonist sulpiride did not prevent the suppressive dopamine effect. The data indicate that dopamine strongly depresses cholinergic gamma oscillations in area CA3 of rat hippocampus by activation of D1-like dopamine receptors and that this effect is most likely mediated via impairment of interneurons involved in generation and maintenance of the carbachol-induced network rhythm.