Drug abuse, brain calcification and glutamate-induced neurodegeneration

Positive and negative reinforcing systems are part of the mechanism of drug dependence. Drugs with abuse potential may change the manner of response to negative emotional stimuli, activate positive emotional reactions and possess primary reinforcing properties. Catecholaminergic and peptidergic processes are of importance in these mechanisms. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction. Presently, glutamate is candidate to play a role in the enduring effects of drugs of abuse. For example, it participates in the chronic pathological changes of corticostriatal terminals produced by methamphetamine. At the synaptic level, a link between over-activation of glutamate receptors, [C(a2+)](i) increase and neuronal damage has been clearly established leading to neurodegeneration. Thus, neurodegeneration can start after an acute over-stimulation whose immediate effects depend on a diversity of calcium-activated mechanisms. If sufficient, the initial insult results in calcification and activation of a chronic on-going process with a progressive loss of neurons. At present, long-term effects of drug dependence underlie an excitotoxicity process linked to a polysynaptic pathway that dynamically regulates synaptic glutamate. Retaliatory mechanisms include energy capability of the neurons, inhibitory systems and cytoplasmic calcium precipitation as part of the neuron-glia interactions. This paper presents an integrated view of these molecular and cellular mechanisms to help understand their relationship and interdependence in a chronic pathological process that suggest new targets for therapeutic intervention.

The Immune-Pineal Axis Stress as a Modulator of Pineal Gland Function

The temporal organization of mammals presents a daily adjustment to the environmental light/dark cycle. The environmental light detected by the retina adjusts the central clock in the suprachiasmatic nuclei, which innervate the pineal gland through a polysynaptic pathway. During the night, this gland produces and releases the nocturnal hormone melatonin, which circulates throughout the whole body and adjusts several bodily functions according to the existence and duration of darkness. We have previously shown that during the time frame of an inflammatory response, pro-inflammatory cytokines, such as tumor necrosis factor-a, inhibit while anti-inflammatory mediators, such as glucocorticoids, enhance the synthesis of melatonin, interfering in the daily adjustment of the light/dark cycle. Therefore, injury disconnects the organism from environmental cycling, while recovery restores the light/dark information to the whole organism. Here, we extend these observations by evaluating the effect of a mild restraint stress, which did not induce macroscopic gastric lesions. After 2 h of restraint, there was an increase in circulating corticosterone, indicating activation of the hypothalamus-pituitary-adrenal (HPA) axis. In parallel, an increase in melatonin production was observed. Taking into account the data obtained with models of inflammation and stress, we reinforce the hypothesis that the activity of the pineal gland is modulated by the state of the immune system and the HPA axis, implicating the darkness hormone melatonin as a modulator of defense responses.

Vibration as a method for muscular stimulation

Human reactions to vibration have been extensively investigated in the past. Vibration, as well as whole-body vibration (WBV), has been commonly considered as an occupational hazard for its detrimental effects on human condition and comfort. Although long term exposure to vibrations may produce undesirable side-effects, a great part of the literature is dedicated to the positive effects of WBV when used as method for muscular stimulation and as an exercise intervention. Whole body vibration training (WBVT) aims to mechanically activate muscles by eliciting neuromuscular activity (muscle reflexes) via the use of vibrations delivered to the whole body. The most mentioned mechanism to explain the neuromuscular outcomes of vibration is the elicited neuromuscular activation. Local tendon vibrations induce activity of the muscle spindle Ia fibers, mediated by monosynaptic and polysynaptic pathways: a reflex muscle contraction known as the Tonic Vibration Reflex (TVR) arises in response to such vibratory stimulus. In WBVT mechanical vibrations, in a range from 10 to 80 Hz and peak to peak displacements from 1 to 10 mm, are usually transmitted to the patient body by the use of oscillating platforms. Vibrations are then transferred from the platform to a specific muscle group through the subject body. To customize WBV treatments, surface electromyography (SEMG) signals are often used to reveal the best stimulation frequency for each subject. Use of SEMG concise parameters, such as root mean square values of the recordings, is also a common practice; frequently a preliminary session can take place in order to discover the more appropriate stimulation frequency. Soft tissues act as wobbling masses vibrating in a damped manner in response to mechanical excitation; Muscle Tuning hypothesis suggest that neuromuscular system works to damp the soft tissue oscillation that occurs in response to vibrations; muscles alters their activity to dampen the vibrations, preventing any resonance phenomenon. Muscle response to vibration is however a complex phenomenon as it depends on different parameters, like muscle-tension, muscle or segment-stiffness, amplitude and frequency of the mechanical vibration. Additionally, while in the TVR study the applied vibratory stimulus and the muscle conditions are completely characterised (a known vibration source is applied directly to a stretched/shortened muscle or tendon), in WBV study only the stimulus applied to a distal part of the body is known. Moreover, mechanical response changes in relation to the posture. The transmissibility of vibratory stimulus along the body segment strongly depends on the position held by the subject. The aim of this work was the investigation on the effects that the use of vibrations, in particular the effects of whole body vibrations, may have on muscular activity. A new approach to discover the more appropriate stimulus frequency, by the use of accelerometers, was also explored. Different subjects, not affected by any known neurological or musculoskeletal disorders, were voluntarily involved in the study and gave their informed, written consent to participate. The device used to deliver vibration to the subjects was a vibrating platform. Vibrations impressed by the platform were exclusively vertical; platform displacement was sinusoidal with an intensity (peak-to-peak displacement) set to 1.2 mm and with a frequency ranging from 10 to 80 Hz. All the subjects familiarized with the device and the proper positioning. Two different posture were explored in this study: position 1 - hack squat; position 2 - subject standing on toes with heels raised. SEMG signals from the Rectus Femoris (RF), Vastus Lateralis (VL) and Vastus medialis (VM) were recorded. SEMG signals were amplified using a multi-channel, isolated biomedical signal amplifier The gain was set to 1000 V/V and a band pass filter (-3dB frequency 10 - 500 Hz) was applied; no notch filters were used to suppress line interference. Tiny and lightweight (less than 10 g) three-axial MEMS accelerometers (Freescale semiconductors) were used to measure accelerations of onto patient’s skin, at EMG electrodes level. Accelerations signals provided information related to individuals’ RF, Biceps Femoris (BF) and Gastrocnemius Lateralis (GL) muscle belly oscillation; they were pre-processed in order to exclude influence of gravity. As demonstrated by our results, vibrations generate peculiar, not negligible motion artifact on skin electrodes. Artifact amplitude is generally unpredictable; it appeared in all the quadriceps muscles analysed, but in different amounts. Artifact harmonics extend throughout the EMG spectrum, making classic high-pass filters ineffective; however, their contribution was easy to filter out from the raw EMG signal with a series of sharp notch filters centred at the vibration frequency and its superior harmonics (1.5 Hz wide). However, use of these simple filters prevents the revelation of EMG power potential variation in the mentioned filtered bands. Moreover our experience suggests that the possibility of reducing motion artefact, by using particular electrodes and by accurately preparing the subject’s skin, is not easily viable; even though some small improvements were obtained, it was not possible to substantially decrease the artifact. Anyway, getting rid of those artifacts lead to some true EMG signal loss. Nevertheless, our preliminary results suggest that the use of notch filters at vibration frequency and its harmonics is suitable for motion artifacts filtering. In RF SEMG recordings during vibratory stimulation only a little EMG power increment should be contained in the mentioned filtered bands due to synchronous electromyographic activity of the muscle. Moreover, it is better to remove the artifact that, in our experience, was found to be more than 40% of the total signal power. In summary, many variables have to be taken into account: in addition to amplitude, frequency and duration of vibration treatment, other fundamental variables were found to be subject anatomy, individual physiological condition and subject’s positioning on the platform. Studies on WBV treatments that include surface EMG analysis to asses muscular activity during vibratory stimulation should take into account the presence of motion artifacts. Appropriate filtering of artifacts, to reveal the actual effect on muscle contraction elicited by vibration stimulus, is mandatory. However as a result of our preliminary study, a simple multi-band notch filtering may help to reduce randomness of the results. Muscle tuning hypothesis seemed to be confirmed. Our results suggested that the effects of WBV are linked to the actual muscle motion (displacement). The greater was the muscle belly displacement the higher was found the muscle activity. The maximum muscle activity has been found in correspondence with the local mechanical resonance, suggesting a more effective stimulation at the specific system resonance frequency. Holding the hypothesis that muscle activation is proportional to muscle displacement, treatment optimization could be obtained by simply monitoring local acceleration (resonance). However, our study revealed some short term effects of vibratory stimulus; prolonged studies should be assembled in order to consider the long term effectiveness of these results. Since local stimulus depends on the kinematic chain involved, WBV muscle stimulation has to take into account the transmissibility of the stimulus along the body segment in order to ensure that vibratory stimulation effectively reaches the target muscle. Combination of local resonance and muscle response should also be further investigated to prevent hazards to individuals undergoing WBV treatments.

Short-Term Plasticity in a Computational Model of the Tail-Withdrawal Circuit in Aplysia.

The tail-withdrawal circuit of Aplysia provides a useful model system for investigating synaptic dynamics. Sensory neurons within the circuit manifest several forms of synaptic plasticity. Here, we developed a model of the circuit and investigated the ways in which depression (DEP) and potentiation (POT) contributed to information processing. DEP limited the amount of motor neuron activity that could be elicited by the monosynaptic pathway alone. POT within the monosynaptic pathway did not compensate for DEP. There was, however, a synergistic interaction between POT and the polysynaptic pathway. This synergism extended the dynamic range of the network, and the interplay between DEP and POT made the circuit responded preferentially to long-duration, low-frequency inputs.

Short-Term Plasticity in a Computational Model of the Tail-Withdrawal Circuit in Aplysia.

The tail-withdrawal circuit of Aplysia provides a useful model system for investigating synaptic dynamics. Sensory neurons within the circuit manifest several forms of synaptic plasticity. Here, we developed a model of the circuit and investigated the ways in which depression (DEP) and potentiation (POT) contributed to information processing. DEP limited the amount of motor neuron activity that could be elicited by the monosynaptic pathway alone. POT within the monosynaptic pathway did not compensate for DEP. There was, however, a synergistic interaction between POT and the polysynaptic pathway. This synergism extended the dynamic range of the network, and the interplay between DEP and POT made the circuit responded preferentially to long-duration, low-frequency inputs.

Mechanisms for the release of dopamine from turtle retina

The retinal circuitry underlying the release of dopamine was examined in the turtle, Pseudemys scripta elegans, using neurochemical release studies, anatomical techniques, and biochemistry. There was a dose- and calcium-dependent release of dopamine from turtle retinas incubated in $\sp3$H-dopamine after perfusion of the GABA antagonist bicuculline. This indicated that dopamine release was tonically inhibited by GABA. Other putative retinal transmitters were examined. Glutamate antagonists selective for hyperpolarizing bipolar cells, such as 2,3-piperidine dicarboxylic acid (PDA), caused dose- and calcium-dependent release of dopamine from the retina. In contrast, release was not observed after perfusion with 4-aminophosphonobutyric acid, a specific antagonist of depolarizing bipolar cells. This indicated that depolarizing bipolar cells were not involved in retinal circuitry underlying the release of dopamine in the turtle retina. The release produced by PDA was blocked by bicuculline, indicating a polysynaptic mechanism of release. None of the other agents tested, which included carbachol, strychnine, dopamine uptake inhibitors, serotonin, tryptamine, muscimol, melatonin, or dopamine itself produced release.^ The cells capable of the release of dopamine were identified using both uptake autoradiography and immunocytochemical localization with dopamine antisera. The simplest circuitry based on these findings is signal transmission from photoreceptors to hyperpolarizing bipolar cells then to GABAergic cells, and finally to dopaminergic amacrine cells. ^

Anatomical organization of the memory underlying sensitization in the siphon-withdrawal reflex circuit of {\it Aplysia californica}

Previous studies have shown that short-term sensitization of the Aplysia siphon-withdrawal reflex circuit results in multiple sites of change in synaptic efficacy. In this dissertation I have used a realistic modeling approach (using an integrate-and-fire scheme), in conjunction with electrophysiological experiments, to evaluate the contribution of each site of plasticity to the sensitized response.^ This dissertation contains a detailed description of methodology for the construction of the model circuit, consisting of the LFS motor neurons and ten interneurons known to convey excitatory input to them. The model replicates closely the natural motor neuron firing response to a brief tactile stimulus.^ The various circuit elements have different roles for producing circuit output. For example, the sensory connections onto the motor neuron are important for the production of the phasic response, while the polysynaptic interneuronal connections are important for producing the tonic response.^ The multiple sites of plasticity that produce changes in circuit output also have specialized roles. Presynaptic facilitation of the sensory neuron to LFS connection enhances only the phasic component of the motor neuron firing response. The sensory neuron to interneuron connections primarily enhance the tonic component of the motor neuron firing response. Also, the L29 posttetanic potentiation and the L30 presynaptic inhibition primarily enhance the tonic component of the motor neuron firing response. Finally, the information content at the various sites of plasticity can shift with changes in stimulus intensity. This suggests that while the sites of plasticity encoding memory are fixed, the information content at these sites can be dynamic, shifting in anatomical location with changes in the intensity of the test stimulus.^ These sites of plasticity also produce specific changes in the behavioral response. Sensory-LFS plasticity selectively increases the amplitude of the behavioral response, and has no effect on the duration of the behavioral response. Interneuronal plasticity (L29 and L30) affects both the amplitude and duration of the behavioral response. Other sensory plasticity also affect both the amplitude and duration of the behavioral response, presumably by increasing the recruitment of the interneurons, which provide all of the effect on duration of the behavioral response. ^

Context-sensitive synaptic plasticity and temporal-to-spatial transformations in hippocampal slices

Hippocampal slices are used to show that, as a temporal input pattern of activity flows through a neuronal layer, a temporal-to-spatial transformation takes place. That is, neurons can respond selectively to the first or second of a pair of input pulses, thus transforming different temporal patterns of activity into the activity of different neurons. This is demonstrated using associative long-term potentiation of polysynaptic CA1 responses as an activity-dependent marker: by depolarizing a postsynaptic CA1 neuron exclusively with the first or second of a pair of pulses from the dentate gyrus, it is possible to “tag” different subpopulations of CA3 neurons. This technique allows sampling of a population of neurons without recording simultaneously from multiple neurons. Furthermore, it reflects a biologically plausible mechanism by which single neurons may develop selective responses to time-varying stimuli and permits the induction of context-sensitive synaptic plasticity. These experimental results support the view that networks of neurons are intrinsically able to process temporal information and that it is not necessary to invoke the existence of internal clocks or delay lines for temporal processing on the time scale of tens to hundreds of milliseconds.

Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster

The ability to tolerate a low-O2 environment varies widely among species in the animal kingdom. Some animals, such as Drosophila melanogaster, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O2, we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O2 deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.

Cortical cell death induced by IL-1 is mediated via actions in the hypothalamus of the rat

The cytokine IL-1 mediates diverse forms of neurodegeneration, but its mechanism of action is unknown. We have demonstrated previously that exogenous and endogenous IL-1 acts specifically in the rat striatum to dramatically enhance ischemic and excitotoxic brain damage and cause extensive cortical injury. Here we tested the hypothesis that this distant effect of IL-1 is mediated through polysynaptic striatal outputs to the cortex via the hypothalamus. We show that IL-1β injected into the rat striatum with the excitotoxin α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (S-AMPA) caused increased expression of IL-1β (mRNA and protein) mainly in the cortex where maximum injury occurs. Marked increases in IL-1β mRNA and protein were also observed in the hypothalamus. S-AMPA, injected alone into the striatum, caused only localized damage, but administration of IL-1β into either the striatum or the lateral hypothalamus immediately after striatal S-AMPA resulted in widespread cell loss throughout the ipsilateral cortex. Finally we showed that the cortical cell death produced by striatal coinjection of S-AMPA and IL-1β was significantly reduced by administration of the IL-1 receptor antagonist into the lateral hypothalamus. These data suggest that IL-1β can act in the hypothalamus to modify cell viability in the cortex. We conclude that IL-1-dependent pathways project from the striatum to the cortex via the hypothalamus and lead to cortical injury, and that these may contribute to a number of human neurological conditions including stroke and head trauma.

BDNF but not NT-4 is required for normal flexion reflex plasticity and function

Neurotrophins can directly modulate the function of diverse types of central nervous system synapses. Brain-derived neurotrophic factor (BDNF) might be released by nociceptors onto spinal neurons and mediate central sensitization associated with chronic pain. We have studied the role of BDNF and neurotrophin-4 (NT-4), both ligands of the trkB tyrosine kinase receptor, in synaptic transmission and reflex plasticity in the mouse spinal cord. We used an in vitro spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents in BDNF- and NT-4-deficient mice. In situ hybridization studies show that both these neurotrophins are synthesized by sensory neurons, and NT-4, but not BDNF, also is expressed by spinal neurons. BDNF null mutants display selective deficits in the ventral root potential (VRP) evoked by stimulating nociceptive primary afferents whereas the non-nociceptive portion of the VRP remained unaltered. In addition, activity-dependent plasticity of the VRP evoked by repetitive (1 Hz) stimulation of nociceptive primary afferents (termed wind-up) was substantially reduced in BDNF-deficient mice. This plasticity also was reduced in a reversible manner by the protein kinase inhibitor K252a. Although the trkB ligand NT-4 is normally present, reflex properties in NT-4 null mutant mice were normal. Pharmacological studies also indicated that spinal N-methyl-d-aspartate receptor function was unaltered in BDNF-deficient mice. Using immunocytochemistry for markers of nociceptive neurons we found no evidence that their number or connectivity was substantially altered in BDNF-deficient mice. Our data therefore are consistent with a direct role for presynaptic BDNF release from sensory neurons in the modulation of pain-related neurotransmission.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.