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Diffusion from an injected volume of a substance in brain tissue with arbitrary volume fraction and tortuosity
Abstract
When a substance is pressure-injected from a micropipette into the extracellular space of the brain it may either form a cavity or it may infiltrate the extracellular space. In either case subsequent diffusion is governed by the volume fraction and tortuosity of the brain tissue as well as the diffusion coefficient of the substance itself. Appropriate equations, solutions and approximations to these problems are discussed. The results are relevant to the interpretation of studies on neuropharmacology and in situ electrochemistry.
... Our histological analysis confirmed the correct targeting of the DRN in experimental mice used in the current study ( Figure 1A,B). Based on the injection volume delivered and the diffusion coefficients, the injected OXA should remain confined to the DRN [64]. A randomly assigned Latin square unblinded design was used to evenly distribute the treatments. ...
... Previous studies have shown that non-DREADD-expressing (control) mice do not show any behavioral or thermogenic effects after either IP CNO or saline (vehicle) injections, whereas DREADD-expressing mice injected with CNO showed changes in behavior [43][44][45] and thermogenesis [93], confirming that the observed effects in the current study are due to specific orexin neuron activation by CNO in DREADD expressing animals. We also did not measure the spread of the microinjected pharmacological agents, but Nicholson's work showing that interaction between the ligand and receptor limits diffusion suggests that it is unlikely that appreciable or effective amounts extend beyond the DRN [64]. Another limitation is the lack of data showing cFos expression following pharmacological manipulation in mice. ...
Orexin/hypocretin terminals innervate the dorsal raphe nucleus (DRN), which projects to motor control areas important for spontaneous physical activity (SPA) and energy expenditure (EE). Orexin receptors are expressed in the DRN, and obesity-resistant (OR) rats show higher expression of these receptors in the DRN and elevated SPA/EE. We hypothesized that orexin-A in the DRN enhances SPA/EE and that DRN-GABA modulates the effect of orexin-A on SPA/EE. We manipulated orexin tone in the DRN either through direct injection of orexin-A or through the chemogenetic activation of lateral-hypothalamic (LH) orexin neurons. In the orexin neuron activation experiment, fifteen minutes prior to the chemogenetic activation of orexin neurons, the mice received either the GABA-agonist muscimol or antagonist bicuculline injected into the DRN, and SPA/EE was monitored for 24 h. In a separate experiment, orexin-A was injected into the DRN to study the direct effect of DRN orexin on SPA/EE. We found that the activation of orexin neurons elevates SPA/EE, and manipulation of GABA in the DRN does not alter the SPA response to orexin neuron activation. Similarly, intra-DRN orexin-A enhanced SPA and EE in the mice. These results suggest that orexin-A in the DRN facilitates negative energy balance by increasing physical activity-induced EE, and that modulation of DRN orexin-A is a potential strategy to promote SPA and EE.
... In a third and separate investigation, microinjection of glutamate was shown to induce either a pressordepressor response and bradycardia (90nl, 200mM) or a single depressor-bradycardic response (90nl, lOOmM), thus suggesting the differences reported are not merely due to the use of various species of animal (Miura & Takayama, 1991). In the light of these reports, the variable results observed by these investigators and the lack of evoked responses in this study may be due to the effects of different anaesthetics on parabrachial neurons or to the problems intrinsic to pressure injections (Nicholson, 1985). ...
... These discrepancies in the spread of injectate may also be indicative of the two scenarios evident upon pressure injection. Either the drug is injected directly into the extracellular space, from where it then diffuses, or the ejection pressure forms a cavity filled with drug, from which it then diffuses (Nicholson, 1985). However, most of the injection sites in the present study were placed at the intermediate level of the PBN, from which kainate has been reported to significantly attenuate the lobule IXb evoked responses in both the anaesthetised and unanaesthetised decerebrate rabbit . ...
The aim of the present study was to ascertain possible roles for the posterior cerebellar vermis in cardiovascular control using the anaesthetised, paralysed and artificially ventilated rabbit. The cortex of lobule IXb was stimulated both electrically and chemically and the effects of removal of lobules VI, VII and IX on the cardiorespiratory responses evoked from defensive behaviour related structures were observed. Arterial blood pressure, heart rate, femoral and renal vascular blood flow, and phrenic and renal nerve activities were routinely measured. Removal of lobule IX resulted in an increase in the sensitivity of the baroreceptor reflex response to a pressor challenge induced by intraluminal balloon inflations in the descending aorta. The increase in baroreflex gain was still evident when the experiments were carried out under β1-receptor blockade, the cell bodies in only lobule IXb were lesioned and whether the gain was calculated using R-R intervals derived from the heart rate or absolute R-R intervals. Stimulation of the HDA or PAG and ACe results in cardiorespiratory responses that are synonymous with those which occur in "fight or flight" and "playing dead" behaviours, respectively. Removal of lobule IX, but not lobules VI and VII, resulted in attenuated HDA, PAG and ACe evoked cardiovascular responses. On the other hand, simultaneous stimulation of lobule IXb with either of these structures resulted in facilitated "cardiovascular defence responses". Indeed, chemical activation of neurons in the HDA, PAG, ACe and lobule IXb identified the structure related nature of these cerebellar-midbrain/forebrain interactions. The cardiovascular effects elicited from the HDA or ACe and lobule IXb were vastly attenuated when cell bodies in the ipsilateral lateral parabrachial nucleus (LPBN) were lesioned with the excitotoxin kainic acid. Neurons in lobule IX demonstrated their ability to receive baroreceptor and hypothalamic inputs upon single or paired-pulse stimulation of the ipsilateral aortic nerve and hypothalamic defence area (HDA). A possible role for lobule IX of the posterior vermis in cardiovascular control is discussed in relation to published physiological and neuroanatomical studies and the results gained in the present study.
... We used microinjection volumes of 70 nl to functionally identify VRC subareas and volumes of 700 nl to achieve maximal effects of glutamate antagonist injections into each VRC subarea. According to Nicholson (Nicholson, 1985), a 70 nl microinjection produces an initial spherical volume with a radius of 429 μm (r = 104.3*V 1/3 μm). ...
... This calculation takes into account that in an isotropic medium extracellular space is 21% of the tissue volume, which results in a larger spherical radius as the injected volume fills the smaller space between cells. In addition, the diffusion path is tortuous, and this effect is accounted for by the tortuosity factor (λ), which has been found to be ∼1.6 (Nicholson, 1985;Nicholson and Syková, 1998). The rate of diffusion is inversely proportional to the square root of the molecular weight (MW). ...
Glutamate is the predominant excitatory neurotransmitter in the ventral respiratory column; however, the contribution of glutamatergic excitation in the individual subregions to respiratory rhythm generation has not been fully delineated. In an adult, in vivo, decerebrate rabbit model during conditions of mild hyperoxic hypercapnia we blocked glutamatergic excitation using the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(–)-2-amino-5-phosphonopentanoic acid (AP5). Disfacilitation of the preBötzinger Complex caused a decrease in inspiratory and expiratory duration as well as peak phrenic amplitude and ultimately apnea. Disfacilitation of the Bötzinger Complex caused a decrease in inspiratory and expiratory duration; subsequent disfacilitation of the preBötzinger Complex resulted in complete loss of the respiratory pattern but maintained tonic inspiratory activity. We conclude that glutamatergic drive to the preBötzinger Complex is essential for respiratory rhythm generation. Glutamatergic drive to the Bötzinger Complex significantly affects inspiratory and expiratory phase duration. Bötzinger Complex neurons are responsible for maintaining the silent expiratory phase of the phrenic neurogram.
... In two additional preparations, bilateral control microinjections of 10 mM ACh/5 mM physostigmine were performed at different medullary locations (4 trials) sufficiently far (> 0.8 mm) from the responsive sites (see e.g. Nicholson, 1985;Lipski et al., 1988;Mutolo et al., 2007Mutolo et al., , 2012Mutolo et al., , 2014Sykova and Nicholson, 2008;Cinelli et al., 2013Cinelli et al., , 2016. With respect to the responsive sites, they were performed into the NTS region 1 mm rostral and 1 mm lateral to the midline (1 trial), into the adjacent reticular formation (2 trials) and into the reticular formation located 1 mm more caudal at the same depth (1 trial). ...
... The distance of control microinjections from the responsive sites derives from previous observations on the spread of the injectate (see e.g. Nicholson, 1985;Lipski et al., 1988;Mutolo et al., 2002aMutolo et al., , 2005Bongianni et al., 2008Bongianni et al., , 2010Sykova and Nicholson, 2008). Given the very short lasting effects of ACh due not only to diffusion, but especially to acetylcholinesterase-induced degradation, we injected in combination 10 mM ACh and 5 mM physostigmine. ...
... In four additional preparations, bilateral control microinjections of 1 mM lisinopril were performed at different medullary locations sufficiently far from the responsive sites Lipski et al., 1988;Mutolo et al., 2007Mutolo et al., , 2008Mutolo et al., , 2009Mutolo et al., , 2012Mutolo et al., , 2014Nicholson, 1985;Sykova and Nicholson, 2008). Four microinjections of 1 mM lisinopril were executed into each of the following neural structures (for comparison, see Meessen and Olszewski, 1949;Shek et al., 1986): the nucleus cuneatus medialis, the nucleus tractus spinalis nervi trigemini and the region located >0.8 mm caudal to the responsive NTS sites. ...
... The specificity of drug-induced effects is supported by the absence of changes in cough-reflex responses induced by bilateral microinjections of the vehicle solution into the caudal NTS or by drug microinjections into regions sufficiently away from the responsive sites. For a more exhaustive appraisal and discussion on the spread of injected drugs see also Lipski et al., 1988;Nicholson, 1985;Sykova and Nicholson, 2008. Interestingly, changes in mean arterial blood pressure that may affect the cough reflex (Poliacek et al., 2011) were not observed. ...
... In two additional preparations, bilateral control microinjections of 10 mM ACh/5 mM physostigmine were performed at different medullary locations (4 trials) sufficiently far (> 0.8 mm) from the responsive sites (see e.g. Nicholson, 1985;Lipski et al., 1988;Mutolo et al., 2007Mutolo et al., , 2012Mutolo et al., , 2014Sykova and Nicholson, 2008;Cinelli et al., 2013Cinelli et al., , 2016. With respect to the responsive sites, they were performed into the NTS region 1 mm rostral and 1 mm lateral to the midline (1 trial), into the adjacent reticular formation (2 trials) and into the reticular formation located 1 mm more caudal at the same depth (1 trial). ...
... The distance of control microinjections from the responsive sites derives from previous observations on the spread of the injectate (see e.g. Nicholson, 1985;Lipski et al., 1988;Mutolo et al., 2002aMutolo et al., , 2005Bongianni et al., 2008Bongianni et al., , 2010Sykova and Nicholson, 2008). Given the very short lasting effects of ACh due not only to diffusion, but especially to acetylcholinesterase-induced degradation, we injected in combination 10 mM ACh and 5 mM physostigmine. ...
We have previously shown that cough potentiation induced by intravenous administration of the AT1 receptor antagonist losartan is lower than that induced by the ACE inhibitor lisinopril in anesthetized and awake rabbits. Since losartan and lisinopril cross the blood-brain barrier, their central action on the cough reflex can be hypothesized. Mechanical stimulation of the tracheobronchial tree and citric acid inhalation were used to induce cough reflex responses in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50nl) of losartan (5mM), lisinopril (1mM), bradykinin (0.05mM), HOE-140 (0.2mM, a bradykinin B2 receptor antagonist) and CP-99,994 (1mM, an NK1 receptor antagonist) were performed into the caudal nucleus tractus solitarii, the predominant site of termination of cough-related afferents. Lisinopril, but not losartan increased the cough number. This effect was reverted by HOE-140 or CP-99,994. Cough potentiation was also induced by bradykinin. The results support for the first time a central protussive action of lisinopril mediated by an accumulation of bradykinin and substance P.
Copyright © 2015. Published by Elsevier B.V.
... Histological control of the pipette track and, in some cases, the localization of fluorescent beads microinjected into the cNTS have been performed also in the present study (Fig. 1). The specificity of drug-induced effects is confirmed by the lack of changes in baseline respiratory activity and in particular, in cough reflex responses following muscarine microinjections at sites sufficiently far (> 0.8 mm) from the responsive sites, as well as following bilateral microinjections of the vehicle solutions into the responsive sites of the cNTS (15,(42)(43)(44). Furthermore, concentrations of agonist and antagonist drugs used in the present study were comparable to those previously used in in vivo preparations (see Methods). ...
... Some works elongate the cross-section to generate 3D geometry [22,45], which means that all the axons share the same longitudinal shape and tortuosity, as shown in Fig. 2(B). This simplification provides a practical solution to this challenging problem and may be valid when modelling of mechanical properties; however, this is unjustified for the solution of the diffusion problem since longitudinal tortuosity of the axons has great impact on the particle's diffusivity [46][47][48]. Some other more sophisticated geometric models also surfer from the similar concerns [49]. ...
Purpose: Brain disorders have become a serious problem for healthcare worldwide. Nanoparticle-based drugs are one of the emerging therapies and have shown great promise to treat brain diseases. Modifications on particle size and surface charge are two efficient ways to increase the transport efficiency of nanoparticles through brain-blood barrier; however, partly due to the high complexity of brain microstructure and limited visibility of Nanoparticles (NPs), our understanding of how these two modifications can affect the transport of NPs in the brain is insufficient.
Methods:
In this study, a framework, which contains a stochastic geometric model of brain white matter (WM) and a mathematical particle tracing model, was developed to investigate the relationship between particle size/surface charge of the NPs and their effective diffusion coefficients (D) in WM.
Results:
The predictive capabilities of this method have been validated using published experimental tests. For negatively charged NPs, both particle size and surface charge are positively correlated with D before reaching a size threshold. When Zeta potential (Zp) is less negative than -10 mV, the difference between NPs’ D in WM and pure interstitial fluid (IF) is limited.
Conclusion:
A deeper understanding on the relationships between particle size/surface charge of NPs and their D in WM has been obtained. The results from this study and the developed modelling framework provide important tools for the development of nano-drugs and nano-carriers to cure brain diseases.
... Table 1). The volumes of our microinjections are very unlikely to have directly modified the activity of neurons at a distance more than 0.5 mm away from the micropipette tip (17,23,(37)(38)(39). ...
Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg i.v. initially). Neuronal excitation by unilateral microinjection of glutamate analog D,L-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared to effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate related neuronal excitation by bilateral microinjections of non-specific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.
... The diffusion of a substance following its brain microinjection depends upon the diffusion coefficient of the drug and the characteristics of the brain region [26]. Interestingly, methylene blue microinjected into the CNS in a 0.2 μl volume diffuses an average ratio of 520 μm [19]. ...
The melanin-concentrating hormone (MCH) is a neuromodulatory peptide present in neurons located in the postero-lateral hypothalamus and incerto-hypothalamic area. These neurons project widely throughout the central nervous system, including the serotonergic dorsal raphe nucleus (DR), a waking-promoting and REM sleep permissive region. Although it is presently accepted that the MCHergic system is involved in the generation of sleep, most studies have been done in rodents, were only the MCH receptor type 1 (MCHR-1) is functionally active. In humans, as well as in carnivores, the MCHR-2 is also a functional receptor. A previous study performed in rats showed that MCH microinjections into the DR induce a significant increase of REM sleep and slow wave sleep (SWS). However, there are no studies on the effect on sleep of MCH applied into the DR of species where both types of MCH receptors are functional.The aim of this work is to confirm the hypothesis that MCH administration into the DR of the cat also promotes sleep. To this purpose, we performed control or MCH (1 µg in 0.2 µl) microinjections into the DR of animals prepared for chronic polysomnographic recordings. Compared to control, MCH produced a significant increase of total sleep time during the first recording hour (43.2 ± 9.5 Vs. 36.6 ± 11.8 min; p < 0.05). In addition, the number of REM sleep episodes was significantly augmented during the first hour and the total recording time (5 h), while SWS latency was decreased (3.5 ± 2.3 Vs. 6.3 ± 4.3 min; p < 0.05). We conclude that the administration of MCH into the DR of the cat promotes sleep.
... Higher concentrations of neuroactive drugs are required with local application due to the exponential decay of drug concentration with the distance from the pipette tip as well as to other factors including the large dilution volume constituted by the extracellular space and the continuous washout in the recording chamber. Microinjection procedures have been extensively discussed in previous reports (Lipski, Bellingham, West, & Pilowsky, 1988;Nicholson, 1985;Nicholson & Sykova, 1998; see e.g. Bongianni, Mutolo, Carfi, & Pantaleo, 2002;Mutolo, Bongianni, Nardone, & Pantaleo, 2005). ...
In mammals, 5‐HTexcitatory respiratory effects imply 5‐HT1A receptor‐mediated disinhibition of preBötzinger complex neurons. In the lamprey, 5‐HT1A receptors are involved in the neural control of locomotion, but their role in the respiratory regulation, particularly at the level of the putative respiratory rhythm generator, the paratrigeminal respiratory group (pTRG), is not known. We hereinvestigatethe respiratory function of inhibitory 5‐HT1A receptorswithin the pTRG of theisolatedbrainstem of the adult lamprey. The 5‐HT1A receptor agonists either bath applied or microinjected into the pTRG did not cause significant effects. However, the selective 5‐HT1A receptor antagonist (S)‐WAY 100135bath applied or microinjected into the pTRG induced depressing respiratory effects or even apnea, thus revealing that 5‐HT exerts a 5‐HT1A receptor‐mediated potent tonic influence on respiration and contributes to maintain baseline levels ofrespiratory activity. Microinjections of strychnine or bicuculline, either alone or in combination, into the pTRGprevented (S)‐WAY 100135‐induced apnea. In addition, immunohistochemical studies corroborate the present findings suggestingthat 5‐HT1A receptors are widely expressed in close apposition to the soma of glycine‐immunoreactive cells located within the pTRG region. The results show that in the lamprey respiratory network 5‐HT exerts a tonic influence on respiration by a potent inhibitory control on both GABAergic and glycinergic mechanisms. The observed disinhibitory effects resemble the excitatory respiratory modulation exerted by 5‐HT1A receptor‐mediated inhibition of glycinergic and/or GABAergic neuronspresent in mammals,supporting the notion that some features of the neuronal network subserving respiratory rhythm generation are highly conserved throughout phylogeny.
... The injected drug is modeled to have initial uniform distribution with concentration C 0 in a sphere of radius r 0 in the brain tissue. With an extracellular space fraction of the brain of α = 0.21 (33), and injected volume of 0.25 μL at 50 μM concentration of drug in a typical pharmacology study, C 0 = 10.5 μM and r 0 = 0.66 mm. With a given diffusion coefficient, the model provides a quantitative prediction of the distribution of drug concentration after wireless microfluidic delivery. ...
Significance
Neuroscience studies using optogenetics have greatly improved our understanding of brain circuits. Advances in the combined use of optogenetics and pharmacology to further probe important neurochemical signals has lagged, however, in large part due to the inconvenience of conventional cannulated approaches, as well as the difficulty in controlling, powering, and manufacturing optofluidic devices that are reliable and scalable for distribution to the neuroscience community. Here, we present a battery-free, wireless, lightweight optofluidic device that allows adjustable infusion rates, hands-free operation, and unlimited power supply, and is compatible with existing near-field communication (NFC) technology. We show that this device not only reproduces optogenetic and pharmacological experiments in vivo but, additionally, allows far greater freedom of movement in small animal models compared to existing, comparable platforms.
... These findings have not been incorporated yet in a mathematical model of gliomas. However, a reaction-diffusion equation for the concentration C of a substance flowing in the ECS that accounts for the two main structural descriptors of the ECS, the volume fraction α and tortuosity λ, has been intensively studied and validated experimentally [50,51,74]: ...
In 2016 the World Health Organization provided the most recent classification of tumors of the central nervous system based on histology, molecular mechanisms, rate of brain invasion, and a soft tissue-type grading system. The classification of various benign and malignant brain tumors can be used as well as improved by the integration of in silico, in vitro and in vivo studies of brain tumors that will ultimately lead to better diagnosis, treatment protocols and outcomes. In this chapter we review some of the modeling approaches proposed in the literature to predict tumor growth and therapeutic outcome.
... Although, we consider here a model with certain simplifying assumptions, nevertheless, this model can be used as a precursor to models of a number of other clinically important problems. The problem of fluid absorption from an injection site into the brain tissue was investigated by Nicholson [24] by considering two possibilities for fluid flow through the tissue. Firstly, that the injected fluid saturates a region of the tissue and then perfuses to the remaining tissue and secondly that a small cavity of injected fluid forms initially followed by the fluid flow through rest of the tissue. ...
In order to understand the interaction between magnetic field and biological tissues in a physiological system, we present a mathematical model of flow-induced deformation in absorbing porous tissues in the presence of a uniform magnetic field. The tissue is modeled as a deformable porous material in which high cavity pressure drives fluid through the tissue where it is absorbed by capillaries and lymphatics. A biphasic mixture theory is used to develop the model under the assumptions of small solid deformation and strain-dependent linear permeability. A spherical cavity formed during injection of fluid in the tissue is used to find fluid pressure and solid displacement as a function of radial distance and time. The governing nonlinear PDE for fluid pressure is solved numerically using method of lines whereas tissue solid displacement is computed by employing trapezoidal rule. The effect of magnetic parameter on fluid pressure, solid displacement and tissue permeability is illustrated graphically.
... Microinjection procedures have been extensively discussed in previous reports (Lipski et al., 1988, Nicholson, 1985, Nicholson and Sykova, 1998; see also e.g. Bongianni et al., 1997Bongianni et al., , 2002Bongianni et al., , 2008Bongianni et al., , 2010Mutolo et al., 2002Mutolo et al., , 2005. ...
Serotonin (5-HT) has been reported to play excitatory effects on respiration by acting on preBötzinger complex (preBötC) neurons in neonatal or juvenile rodents. However, whether its action is circumscribed to the preBötC and present in other animal species, particularly in adult preparations, is unknown. We investigated the respiratory role of 5-HT within the preBötC and neighbouring respiration-related regions. Experiments were performed on α-chloralose-urethane anesthetized, vagotomized, paralyzed and artificially ventilated rabbits making use of bilateral microinjections (30-50 nl). 5-HT caused excitatory effects on respiratory activity only when applied to the preBötC. These effects were mediated by 5-HT1A and 5-HT3 receptors as shown by microinjections of specific agonists of the different types of 5-HT receptors. Unexpectedly, the blockade of 5-HT1A receptors by methysergide or the specific antagonist (S)-WAY 100135 induced excitatory respiratory effects. Microinjections of the 5-HT3 receptor antagonist ondansetron did not influence respiration, but prevented (S)-WAY 100135-induced responses. The blockade of GABAA receptors by bicuculline within the preBötC prevented the effects of the 5-HT1A receptor agonist 8-OH-DPAT. The involvement of GABAergic inhibition and 5-HT1A receptor-mediated disinhibition is also corroborated by immunohistochemical data. The results show for the first time in an adult animal preparation that 5-HT plays a pivotal role in the modulation of the preBötC activity probably via both presynaptic and postsynaptic mechanisms and highlight the importance of disinhibition phenomena. Present findings may be relevant to some respiratory disorders in which an impairment of central 5-HT mechanisms has been reported, such as sleep apnoea and sudden infant death syndrome.
... An interval of 25 min between microinjections was provided to eliminate any residual effects of the previous injection. The point of the injection site and its three dimensional spread (Edeline, Hars, Hennevin, & Cotillon, 2002;Nicholson, 1985) were assessed by rhodamine microspheres staining of the site and comparison made against the stereotaxic co-ordinate plots based on rat atlas (Paxinos & Watson, 2007). ...
The nucleus retroambiguus (NRA) is a neuronal cell group in the medullary ventrolateral tegmentum, rostrocaudally between the obex and the first cervical spinal segment. NRA neurons are premotor interneurons with direct projections to the motoneurons of soft palate, pharynx and larynx in the nucleus ambiguus in the lateral medulla as well as to the motoneurons in the spinal cord innervating diaphragm, abdominal, and pelvic floor muscles and the lumbosacral motoneurons generating sexual posture. These NRA premotor interneurons receive very strong projections from the periaqueductal gray (PAG) in the context of basic survival mechanisms as fight, flight, freezing, sound production and sexual behavior. In the present study in rat we investigated the physiological motor patterns generated by NRA neurons, as the result of vagal, peripheral chemosensory, and nociceptive stimulation. The results show that the NRA contains phasic respiratory modulated neurons, as well as non-phasic tonically modulated neurons. Stimulation in the various rostrocaudal levels of the NRA generates site-specific laryngeal, respiratory, abdominal and pelvic floor motor activities. Vagal and peripheral chemosensory stimulation induces both excitatory and inhibitory modulation of phasic NRA-neurons, while peripheral chemosensory and nociceptive stimulation causes excitation and inhibition of non-phasic NRA-neurons. These results are in agreement with the concept that the NRA represents a multifunctional group of neurons involved in the output of the emotional motor system, such as vomiting, vocalization, mating, and changes in respiration. This article is protected by copyright. All rights reserved.
... This rationale is based on diffusion coefficients of the injection volume delivered. 28 All cannulae were correctly placed ( Figure 1). ...
Background:
Identifying whether components of total EE are affected by orexin receptor (OXR1 and OXR2) stimulation or antagonism with dual orexin receptor antagonists (DORAs) has relevance for obesity treatment. Orexin receptor stimulation reduces weight gain by increasing total energy expenditure (EE) and EE during spontaneous physical activity (SPA).
Objective:
The purpose of this study was to determine if a DORA (TCS-1102) in the ventrolateral preoptic area (VLPO) reduced orexin-A-induced arousal, SPA, total EE and EE during sleep, rest, wake and SPA and whether the DORA alone reduced total EE and its components. We hypothesized that: (1) a DORA would reduce orexin-A induced increases in arousal, SPA, components of total EE, reductions in sleep and the EE during sleep and (2) the DORA would reduce basal (non-stimulated) SPA and total EE.
Subjects/methods:
Sleep, wakefulness, SPA, and EE were determined after microinjection of the DORA (TCS-1102) and orexin-A in the VLPO of male Sprague-Dawley rats with a unilateral cannula targeted towards the VLPO. Individual components of total EE were determined based on time-stamped data.
Results:
The DORA reduced orexin-A-induced increases in arousal, SPA, total EE, and EE during SPA, wake, rest and sleep 1-h post-injection (P<0.05). Orexin-A significantly reduced sleep and significantly increased EE during sleep 1-h post-injection (P<0.05). Furthermore, the DORA alone significantly reduced total EE and resting EE 2-h post-injection (P<0.05).
Conclusions:
These data suggest that orexin-A reduces weight gain by stimulating total EE through increases in EE during SPA, rest, and sleep. Residual effects of the DORA alone include decreases in total EE and EE during sleep and rest, which may promote weight gain.International Journal of Obesity accepted article preview online, 10 April 2017. doi:10.1038/ijo.2017.92.
... The placement of cannula was deemed to be incorrect if the actual injection location was more than 0.25 mm away from the exact targeted site. The rationale of this criterion is, in part, due to diffusion coefficients of the injection volume delivered (Nicholson, 1985). To do the final analyses of data, incorrectly placed cannula were omitted (Fig. 1). ...
Introduction:
Recent studies have shown that medial pre-optic area (MPOA) of hypothalamus are involved in nociception. Orexin A (hypocretin 1) has been found to have numerous applications including pain modulation. However, the role of orexin A receptors in the MPOA on the nociception has not been yet studied. Therefore, the aim of the present study is to investigate the effect of orexin A microinjection on MPOA on the nociception transmission and morphine induced analgesia in adult male rats.
Methods:
Using stereotaxic surgery, a cannula was implanted at a site 1mm above the MPOA in the anesthetized rats. After the recovery period, tail-flick (TF) latency was measured as 0, 15, 30, 45 and 60min following the onset of two experimental protocols. Two experiments were carried out. Experiment 1: The male rats received intra-MPOA of 25, 100, 1000, 10000pmol/0.5μl orexin A or 0.5μl of aCSF (control, just 5min before the TF assay. Experiment 2: The aim of this experiment was to examine the effect of orexin microinjection into MPOA on morphine analgesia (3mg/kg,s.c). Morphine was administered 30min before orexin A intra-MPOA microinjection (four doses similar to experiment 1) or aCSF, then TF latency was measured.
Results:
The results indicated that microinjection of orexin A into the MPOA showed anti-nociceptive effect in a time-dependent manner. Dose response curve results also revealed that the maximum effective dose of orexin A injection into MPOA for pain inhibition is 1000pmol/0.5μl. Co-administration of systemic morphine and orexin into the MPOA has additive analgesia with different time course compared morphine or orexin alone.
Conclusion:
It can be concluded that MPOA OrexinA receptors play an important role in the modulation of pain in normal and morphine treated male rats.
... Microinjection procedures, their reliability, and the spread of the injectate have been extensively discussed in previous reports (21,(51)(52)(53)(54)(55); see also Refs. 44,59,70). Injection sites were selected by using stereotaxic coordinates according to the atlas of Meessen and Olszewski (48). Their localization was confirmed by the histological control. ...
Cough-related sensory inputs from rapidly-adapting receptors (RARs) and C-fibres are processed by second-order neurons mainly located in the caudal nucleus tractus solitarii (NTS). Both GABAA and glycine receptors have been proven to be involved in the inhibitory control of second-order cells receiving RAR projections. We investigated the role of these receptors within the caudal NTS in the modulation of the cough reflex induced either by mechanical or chemical stimulation of the tracheobronchial tree in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50 nl) of the receptor antagonists bicuculline and strychnine as well as of the receptor agonists muscimol and glycine were performed. Bicuculline (0.1 mM) and strychnine (1 mM) caused decreases in peak abdominal activity and marked increases in respiratory frequency due to decreases in both inspiratory time (TI) and expiratory time (TE), without concomitant changes in arterial blood pressure. Noticeably, these microinjections induced potentiation of the cough reflex consisting of increases in the cough number associated with decreases either in cough-related TI after bicuculline or in both cough-related TI and TE after strychnine. The effects caused by muscimol (0.1 mM) and glycine (10 mM) were in the opposite direction to those produced by the corresponding antagonists. The results show that both GABAA and glycine receptors within the caudal NTS mediate a potent inhibitory modulation of the pattern of breathing and cough reflex responses. They strongly suggest that disinhibition is one important mechanism underlying cough regulation and possibly provide new hints for novel effective antitussive strategies.
... A cannula was deemed incorrect if the actual injection site was farther than 0.25 mm away from the targeted site. This rationale is based on diffusion coefficients of injection volume delivered [25]. In studies 1 and 2, data from animals with incorrectly placed cannulas were excluded from the final analysis, and the number of rats listed in the specific experimental protocols represent the number of rats in the final analysis (all cannulas correctly placed). ...
... Limitations of intracranial microinjection as an approach for elucidating the behavioral state-related roles of neurotransmitter receptors have been reviewed (Myers, 1966;Routtenberg, 1972;Nicholson, 1985). The small size of the mouse brain presents particular challenges for the microinjection approach because distances between structures are short and microinjected drugs diffuse away from their injection sites into neighboring brain regions (Edeline et al., 2002). ...
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and many drugs used to produce sleep, sedation, or general anesthesia enhance transmission at GABA-A receptors (GABA-ARs). The mechanisms underlying these effects remain unknown. The pontine reticular nucleus, oral part (PnO) plays an important role in generating cortical activation and rapid eye movement sleep (REM). GABAergic transmission causes brain-region specific effects on sleep and wakefulness, and in the PnO GABA promotes wakefulness. This dissertation research sought to elucidate the mechanisms by which GABAergic transmission in the PnO of C57BL/6J mouse modulates arousal states.
Aim 1 tested the hypothesis that GABA-ARs in the PnO modulate two phenotypes of arousal ??? sleep/wake states and activity of the cortical electroencephalogram (EEG). PnO microinjection of the GABA-AR agonist muscimol caused a concentration-dependent, reversible increase in wakefulness and decrease in non-REM (NREM) and REM that was blocked by co-administration of the GABA-AR antagonist bicuculline. Muscimol also increased EEG delta power (0.5-4 Hz) during wakefulness and theta power (4-9 Hz) during REM. Microinjection of bicuculline alone caused a concentration dependent decrease in wakefulness and increase in NREM and REM.
Cholinergic transmission in the PnO also contributes to arousal state control, and Aim 2 tested the hypothesis that GABAergic transmission in the PnO modulates acetylcholine (ACh) release in the PnO, breathing rate, and anesthesia recovery time. Microdialysis delivery of bicuculline to the PnO caused a concentration dependent increase in ACh release within the PnO, a decrease in respiratory rate, and an increase in anesthesia recovery time.
These data are the first to characterize the effects of GABAergic transmission on arousal states in mouse PnO. The results from Aim 1 and 2 demonstrate that GABA-ARs in the PnO of C57BL/6J mouse modulate sleep/wake states, activity of the cortical EEG, release of ACh in the PnO, respiratory rate, and anesthesia recovery time. These results support the conclusion that GABA-ARs in mouse PnO promote wakefulness and suggest that increasing GABAergic transmission in the PnO may be one mechanism underlying the phenomenon of paradoxical behavioral activation by some benzodiazepines.
... A limitation of the present study is the difficulty in determining the precise size of injection. As indicated previously (Holden et al., 2014;Jeong et al., 2012), many factors contribute to injection size, including the features of the solution, injector tip size, and neuroanatomical features of the injection site (Lum, Nguyen, & Felpel, 1984;Myers & Hoch, 1978;Nicholson, 1985;Sakai, Swartz, & Woody, 1979). Our smaller injection size and the relatively hydrophilic nature of carbachol limit the average spread of injection (Grossman & Stumpf, 1969;Martin, Knuepfer, & Westfall, 1991;Myers & Hoch, 1978). ...
No evidence to date shows that lateral hypothalamic (LH) stimulation produces orexin-A-mediated antinociception in the spinal cord dorsal horn (SCDH) in a model of neuropathic pain. We conducted experiments to examine the effect of orexin-A-mediated LH stimulation in female rats with chronic constriction injury (CCI) on thermal hyperalgesia. Rats receiving carbachol into the LH demonstrated antinociception on both the left CCI and right nonligated paws (p < .05). Rats were given carbachol in the LH followed by intrathecal injection of the orexin-1 (OX1) receptor antagonist SB-334867, which blocked LH-induced antinociception compared with control groups (p < .05) in the left paw, but not in the right paw. These findings support the hypothesis that LH stimulation produces antinociception in rats with thermal hyperalgesia from neuropathic pain via an orexin-A connection between the LH and the SCDH. Identification of this pathway may lead to studies using orexins to manage clinical pain.
... First, the variability in the injection of 4-AP: although the injection site was carefully chosen, there is inherent variability in the exact position and depth due to the manual positioning. Furthermore, diffusion of the injected volume will vary due to tortuosity of the brain tissue in each mouse (Nicholson, 1985). These limitations may explain the fact that only five out of nine mice presented seizure-like activity. ...
The aim of this thesis is to apply functional connectivity in a variety of animal models, using several optical imaging modalities. Even at rest, the brain shows high metabolic activity: the correlation in slow spontaneous fluctuations identifies remotely connected areas of the brain; hence the term “functional connectivity”. Ongoing changes in spontaneous activity may provide insight into the neural processing that takes most of the brain metabolic activity, and so may provide a vast source of disease related changes.
Brain hemodynamics may be modified during disease and affect resting-state activity. The thesis aims to better understand these changes in functional connectivity due to disease, using functional optical imaging.
The optical imaging techniques explored in the first two contributions of this thesis are Optical Imaging of Intrinsic Signals and Laser Speckle Contrast Imaging, together they can estimate the metabolic rate of oxygen consumption, that closely parallels neural activity. They both have adequate spatial and temporal resolution and are well adapted to image the convexity of the mouse cortex. In the last article, a depth-sensitive modality called photoacoustic tomography was used in the newborn rat. Optical coherence tomography and laminar optical tomography were also part of the array of imaging techniques developed and applied in other collaborations.
The first article of this work shows the changes in functional connectivity in an acute murine model of epileptiform activity. Homologous correlations are both increased and decreased with a small dependence on seizure duration. These changes suggest a potential decoupling between the hemodynamic parameters in resting-state networks, underlining the importance to investigate epileptic networks with several independent hemodynamic measures.
The second study examines a novel murine model of arterial stiffness: the unilateral calcification of the right carotid. Seed-based connectivity analysis showed a decreasing trend of homologous correlation in the motor and cingulate cortices. Graph analyses showed a randomization of the cortex functional networks, suggesting a loss of connectivity, more specifically in the motor cortex ipsilateral to the treated carotid; however these changes are not reflected in differentiated metabolic estimates. Confounds remain due to the fact that carotid rigidification gives rise to neural decline in the hippocampus as well as unilateral alteration of vascular pulsatility; however the results support the need to look at several hemodynamic parameters when imaging the brain after arterial remodeling.
The third article of this thesis studies a model of inflammatory injury on the newborn rat. Oxygen saturation and functional connectivity were assessed with photoacoustic tomography. Oxygen saturation was decreased in the site of the lesion and on the cortex ipsilateral to the injury; however this decrease is not fully explained by hypovascularization revealed by histology. Seed-based functional connectivity analysis showed that inter-hemispheric connectivity is not affected by inflammatory injury.
... 6 the designed values of relative increase of a dose in a biological tissue are instanced due to presence at it a high-Z elements at affecting photons of various energies. Taking into account the pharmacological inertness of the drug Dipentast, the model of diffusion by Nicholson (1985) [13] was taken as the basis of its pharmacokinetic model for the intratumoral administration. These results show that in technology PCT it is possible to reach more localized energy release, than in NCT, at the identical therapeutic ratio. ...
It is well known that application of 10-and Gd-containing drugs in neutron capture therapy (NCT) provides essential increase of local energy release, when the target area is irradiated with thermal neutrons. During the last time we are developing the photon capture therapy (PCT) technology. The physical principle of PCT consists in the increase of local energy release caused by the electrons of photo absorption and the accompanying Auger-cascade on atoms high-Z elements (Gd, Pt, Bi, etc.), which are present into the structure of special drugs at an irradiation of tumor low-energy photons. In both cases efficiency of the method determine not by only irradiation beam aiming on a tumor but also by selective concentration of a drug in a tumor cells or a target volume. Both NCT and PCT are binary technologies and their physical essence has a lot of similarities, keeping in mind a possibility of a local increasing of energy release particular in a clinical target area-tumor. Sometimes methods of a drug delivery into a tumor and dosimeter planning procedures in both cases are the same. Because of all above-both NCT and PCT can be united under a one common term dose-supplementary therapy .
We investigated the hypothesis that the motor trigeminal nucleus, consisting of expiratory motoneurons, might be influential in termination of inspiration. We addressed the issue by comparing the effects on neural respiration of a reversible, unilateral, pharmacologic blockade of the motor trigeminal nucleus (5M), the medial parabrachial nucleus (PB), and of other nearby structures that are neutral for respiration in anesthetized, vagotomized, paralyzed, and ventilated cats. The blockade was achieved by microinjections of 2% xylocaine, laced with Pontamine Sky Blue to identify sites of injections, from the tip of a penetrating microelectrode. Integrated phrenic neurograms were recorded to quantify the time of neural inspiration (TI), expiration (TE), and the peak phrenic amplitude. We found that blockade of the 5M caused a pattern of apneustic respiration, consisting of a selective prolongation of inspiratory phases that were interrupted by short expiratory pauses. In contrast, blockade of the PB resulted in a prolongation of both TI and TE, which corresponds to a mere slowing of respiration. The results confirmed important functions of the rostral pons in ventilatory control but pointed to the 5M rather than PB as a structure underlying the inspiratory off-switch. We conclude that the motor trigeminal nucleus may have a part in the pontine pneumotaxic mechanism.
Orexin-A (OXA) is a hypothalamic neuropeptide implicated in the regulation of wakefulness, appetite, reward processing, muscle tone, motor activity, and other physiological processes. The broad range of systems affected stems from the widespread projections of orexin neurons toward multiple brain regions regulating numerous physiological processes. Orexin neurons integrate nutritional, energetic, and behavioral cues and modulate the functions of target structures. Orexin promotes spontaneous physical activity (SPA), and we recently showed that orexin injected into the ventrolateral preoptic area (VLPO) of the hypothalamus increases behavioral arousal and SPA in rats. However, the specific mechanisms underlying the role of orexin in physical activity are unknown. Here we tested the hypothesis that OXA injected into the VLPO alters the oscillatory activity in the electroencephalogram (EEG) to reflect an increased excitability of the sensorimotor cortex, which may explain the associated increase in SPA. The results showed that OXA increased wakefulness following injections into the VLPO. In addition, OXA altered the power spectrum of the EEG during the awake state by decreasing the power of 5-19 Hz oscillations and increasing the power of >35 Hz oscillations, which are markers of increased sensorimotor excitability. Consistently, we found that OXA induced greater muscle activity. Furthermore, we found a similar change in power spectrum during slow-wave sleep, which suggests that OXA altered the EEG activity in a fundamental way, even in the absence of physical activity. These results support the idea that OXA increases the excitability of the sensorimotor system, which may explain the corresponding increase in awake time, muscle tone, and SPA.
It has been shown that muscarinic acetylcholine receptors (mAChRs) located within the caudal nucleus tractus solitarii (cNTS) mediate a cholinergic inhibitory control mechanism of the cough reflex. Thus, identification of the involved mAChR subtypes could be of considerable interest for novel therapeutic strategies. In pentobarbital sodium-anesthetized, spontaneously breathing rabbits we investigated the contribution of different mAChR subtypes in the modulation of mechanically- and chemically-induced cough reflex. Bilateral microinjections of 1 mM muscarine into the cNTS increased respiratory frequency and decreased expiratory activity even to the complete suppression. Interestingly, muscarine induced strong cough-suppressant effects up to the complete abolition of the reflex. Microinjections of specific mAChR subtype antagonists (M1-M5) into the cNTS were performed. Only microinjections of the M4 antagonist tropicamide (1 mM) prevented muscarine-induced changes in both respiratory activity and cough reflex. The results are discussed in light of the notion that cough involves the activation of the nociceptive system. They also suggest that M4 receptor agonists may have an important role in cough downregulation within the cNTS.
Melanin concentrating hormone (MCH) is a sleep-promoting neuromodulator synthesized by neurons located in the postero-lateral hypothalamus and incerto-hypothalamic area. MCHergic neurons have widespread projections including the serotonergic dorsal (DR) and median (MnR) raphe nuclei, both involved in the control of wakefulness and sleep. In the present study, we explored in rats the presence of the MCH receptor type 1 (MCHR-1) in serotonergic neurons of the MnR by double immunofluorescence. Additionally, we analyzed the effect on sleep of MCH microinjections into the MnR. We found that MCHR-1 protein was present in MnR serotonergic and non-serotonergic neurons. In this respect, the receptor was localized in the primary cilia of these neurons. Compared with saline, microinjections of MCH into the MnR induced a dose-related increase in REM sleep time, which was related to a rise in the number of REM sleep episodes, associated with a reduction in the time spent in W. No significant changes were observed in non-REM (NREM) sleep time. Our data strongly suggest that MCH projections towards the MnR, acting through the MCHR-1 located in the primary cilia, promote REM sleep.
Cough is a very important motor act that may be voluntary, behavioral or simply reflex. Cough shares many similarities with pain at both peripheral and central levels. It shows several components (sensory-discriminative, affective, reflex) that reach their maximum complexity in awake animals and especially in humans who display even placebo/nocebo effects. This chapter is primarily focused on reflex cough triggered by nociceptive stimulation of the tracheobronchial tree and mediated by brainstem neuronal mechanisms. Cough-related inputs are conveyed to the caudal nucleus tractus solitarii (cNTS) that in turn projects to the brainstem respiratory network, that is reconfigured to generate the cough motor pattern. Two medullary structures proved to be strategic sites of action for centrally active drugs, i.e. the cNTS and the caudal ventral respiratory group. Drugs microinjected into these structures cause downregulation or upregulation of the cough reflex. Studies on the basic neural mechanisms subserving the cough reflex may provide hints for further cough researches or novel therapeutic approaches.
The role of the different components of the respiratory network in the mediation of opioid-induced respiratory depression is still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of bilateral microinjections (30-50 nl) of the µ-opioid receptor agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO). Dose-dependent effects were observed. In the preBötC and the BötC 0.1 mM DAMGO microinjections caused mainly reductions in peak phrenic amplitude associated with tonic phrenic activity and irregular (ataxic) patterns of breathing that were more pronounced in the preBötC. Apneic effects developed at 0.5 mM. In the iVRG DAMGO provoked decreases in amplitude and frequency of phrenic bursts at 0.1 mM and apnea at 0.5 mM. Local 5 mM naloxone reversed the apneic effects. The results imply that different components of the respiratory network may contribute to opioid-induced respiratory disorders.
Freshly removed from the skull, the human brain looks like a cauliflower with the consistency of a crème caramel flan. This mundane object conceals the most complex structure ever discovered. When the interior of the brain is examined with a resolution of a few micrometers, using appropriate staining techniques and light microscopy, it is seen to be composed of a vast number of cells. The full complexity of the ensemble of cells is only finally revealed at the submicron level using electron microscopy. This chapter will show how the application of methods and models based on diffusion can lead to an understanding of how brain cells pack together and some of the remarkable properties of the narrow spaces that separate them.
The effects of the dopamine (DA) D1 and D2 receptor agonists SKF38393, bromocriptine and quinpirole, respectively, on spontaneous sleep were analyzed in adult rats prepared for chronic sleep recordings. Local administration of the DAergic agonists into the dorsal raphe nucleus (DRN) during the light phase of the light-dark cycle induced a significant reduction of rapid-eye movement sleep (REMS) and the number of REM periods. Additionally, bromocriptine and quinpirole significantly increased wakefulness (W). Opposite, the microinjection into the DRN of the DA D1 and D2 receptor antagonists SCH23390 and sulpiride, respectively, significantly augmented REMS and the number of REM periods. Pretreatment with SCH23390 and sulpiride prevented the effects of SKF38393 and bromocriptine, respectively, on sleep variables. Our results tend to indicate that DAergic neurons located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) contribute to the regulation of predominantly W and REMS by DRN serotonergic neurons.
This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 µm away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.
The dense aggregation of cells that make up the brain and spinal cord has always prompted discussion about the nature of the interstitial region. This region was termed the “extracellular space” and for many years its extent, and even its existence, was widely disputed. Later the concept of the extracellular space became extended and deepened, primarily because of Schmitt and Samson (1969), who recognized that the space was not merely a saline-filled gap between cells, but rather that it was a complex region defined by the membranes of the bounding cells. These membranes were adorned with a rich variety of long-chain proteoglycans that extended into the extracellular space, potentially endowing it with unusual properties. In recognition of potential functional importance of this domain, the term “brain cell microenvironment,” henceforth abbreviated to BCM, was introduced (Schmitt and Samson, 1969).
: Although medullary control of blood pressure (BP) has been extensively studied, the contribution of critical regions, such as pressor sites in the caudal medulla and upper cervical spinal cord and the lateral tegmental field, remains controversial and underappreciated. A series of pressor sites caudal to the caudal ventrolateral medulla (CVLM), including the caudal pressor area (CPA) and medullocervical pressor area, play an important role in control of BP. Activation and inhibition of these sites elicits pressor and depressor responses, respectively. Basal sympathetic tone is provided principally by the medullary lateral tegmental field and rostral ventrolateral medulla (RVLM). RVLM presympathetic neurons, which project to and drive preganglionic sympathetic somata in the intermediolateral cell column, are powerfully regulated by neurons in CVLM via tonic and phasic inhibition. The current state of knowledge is summarized thus: rostrocaudally organized columns of pressor sites caudal to CVLM extend to the upper cervical spinal cord; CPA pressor responses are RVLM-dependent; CPA mediates pressor responses by (first) inhibiting RVLM-projecting inhibitory CVLM units and (second) activating RVLM-projecting excitatory CVLM units; the chemoreflex is CPA-dependent; the baroreflex is CPA-independent; pressor responses to raphe obscurus stimulation are CPA-dependent; and medullocervical pressor area pressor responses are RVLM-independent, likely mediated by direct projections to the intermediolateral cell column. In this review, we seek to underscore and characterize the critical role played by the caudal medulla and upper cervical spinal cord in BP regulation and highlight important gaps in knowledge in interactions between the caudal medulla and other regions controlling BP, which may prove critical in revealing central mechanisms underlying pathophysiology of, and pharmacotherapeutic targets for, hypertension.
Gd-NCT dosimetry requires exact analysis of the gadolinium
amount in the irradiated target. For this purpose fast and
convenient method for radiographic visualization of
gadolinium-containing preparation (Magnevist) was
developed. By using this metod, the Magnevist
pharmacokinetics was carried out after intratumoral
injection in mice and intramuscular injection in rats.
Obtained data allowed the semiquantitative estimation of
gadolinium amount in injection site. The values of
gadolinium amount in irradiated region depending on time
was used for correcting definition of absorbed dose.
The sympathetic nervous system maintains and regulates arterial pressure and tissue perfusion, via control of cardiac output and vasomotor tone. Sympatho-vascular-mediated increases in blood pressure are effected by arterioloconstriction, which causes an increase in afterload, and/or venoconstriction, which increases venous return, left ventricular preload, and consequently, the force of cardiac contraction via Frank-Starling mechanisms; withdrawal of sympathetic drive elicits reciprocal effects. Spinalization reduces mammalian arterial pressure to 40-50 mm Hg consequent to the elimination of descending medullary pre-sympathetic bulbospinal drive to preganglionic sympathetic fibers in the intermediolateral cell column of the spinal cord. Beyond agreement that sympathetic tone is generated supraspinally, there is only controversy. One hypothesis posits that pre-sympathetic medullary regions, such as the rostral ventrolateral medulla (RVLM) and caudal raph group, possess intrinsic tonic activity. Alternatively, pre-sympathetic medullary regions may receive tonic excitation from other areas in the brainstem. Neurons in the lateral tegmental field (LTF), an exclusively propriobulbar entity (cf. pre-Btzinger complex the propriobulbar inspiratory rhythmogenic kernel of the respiratory network), fire before and project to pre-sympathetic units in RVLM and caudal raph and exhibit activity correlated to the cardiac-related rhythm in sympathetic nerve discharge, making the LTF a likely candidate for the primary source of basal sympathoexcitation. The LTF is additionally involved in a variety of cardiovascular and sympathetic reflexes (i.e., baroreflex, Bezold-Jarisch reflex). As it receives descending afferents from the infralimbic cortex and associated limbic structures, suggesting a role in the sympathetic response to fear, as well as vestibular inputs, consistent with a role in coordinating the sympathetic response with emesis proper, the LTF appears to play an extensive integrative role. In this review, we discuss the LTF, a once mysterious, poorly-characterized, and ill-defined region, the contribution of which to cardiovascular reflexes and basal sympathoexcitation has been more thoroughly elucidated in recent years and any model of central control of sympathetic output must take into consideration the contribution of this important region.
The brain is organized into the cerebrum, brain stem, and cerebellum. The cerebrum consists of two cerebral hemispheres, basal ganglia, and the diencephalon. The hemispheres contain the cerebral cortex and underlying white matter, and are associated with higher order functioning, including memory, cognition, and fine motor control. The basal ganglia, contained within the hemispheres, controls gross motor function. The diencephalon is much smaller than the cerebrum, contains the thalamus and hypothalamus, and is associated with relaying sensory information and controlling the autonomic nervous system. The brainstem contains the mesencephalon, pons and the medulla oblangata. The smallest segment of the brain, the mesencephalon, is located below the diencephalon and is thought to play a role in consciousness. Muscle activation, tone and equilibrium is controlled in the pons and cerebellum located below the mesencephalon, and respiratory and cardiac processes are governed by the medulla oblongata, located directly beneath the pons.
A quantitative evaluation of the thresholds of changes in the firing rate/pattern and depolarizing block of the neuron and the bradycardiac response by pressure microinjection of 10 mM glutamate (Glu) into the region of the nucleus ambiguus (NA) of the ventral medulla was performed in anesthetized rats. A change in neuronal activity was shown with injection of about 2 pmol of Glu. A depolarizing block of single-unit activity could be observed at 2.9 +/- 0.3 nl (similar to30 pmol, n = 22). Maximal bradycardiac response (-50 +/- 5%) was elicited with 4.4 +/- 0.7 nl (similar to50 pmol, n = 10), which is significantly smaller than the ranges used in previous studies. Based on these results, a safe and effective use of 10 mM Glu to induce neuronal or physiological response should be in the range of a few nanoliters and less than 100 pmol, especially for the NA.
Many therapies for drug delivery to the brain are based on diffusion, and diffusion in this extracellular space is based on micro-techniques that can be modelled with classical differential equations such as the point source diffusion equation. In this paper an energy function is constructed using a finite-difference approximation to the governing diffusion equation and then minimized by a Hopfield neural network. The synergy of Hopfield neural networks with finite difference approximation is promising. The neural network approach is capable of giving insight to the complex brain activity better than any other classical numerical method and the parallelism nature of the Hopfield neural networks approach is easier to implement on fast parallel computers and this will make them faster than the traditional methods for modelling this complex problem. Moreover, the effect of the involved parameters on the diffusion distribution and drug delivery in the ECS is investigated.
It has been shown that activation of type 1 orexinergic receptors (ORX1) in several parts of the hypothalamus stimulate food intake. Orexin A receptive sites for food intake exist primarily in a narrow band of the hypothalamus that is known to be involved in control of energy homeostasis. The present study aimed to investigate the role of orexin receptors in the medial preoptic area (MPOA) on food intake in rats. Twenty-four male rats weighing 200-250 g were divided into three groups (n = 8 in each group). Rats were cannulated using stereotaxic coordinates above the MPOA. Normal saline was microinjected into the MPOA in the control group. Another group received intra MPOA microinjection of SB334867, a selective antagonist for ORX1 receptors. In the other group, orexin A was microinjected (0.5 μl of 1 μmol) into the MPOA. Food intake was measured in metabolic cages. The statistical significance of differences between groups was detected by a one way ANOVA. A value of p < 0.05 was considered significant. There was no significant difference in food consumption between saline and SB334867 treated groups. However, activation of the orexin receptor in the MPOA significantly increased food intake during the 2 and 8 h after orexin A microinjection. Our results showed that during ad libitum access to food, activation but not blockade of the MPOA ORX1 receptor can increase food intake in a time-dependent manner. The role of these receptors in hunger and appetite stimulation requires further study.
Copyright © 2015. Published by Elsevier Ireland Ltd.
Convection-enhanced delivery (CED), or also referred to as ‘‘intracerebral clysis'’ and ‘‘highflow microinfusion," is a means of localized drug delivery to the central nervous system. The therapeutic agent, infused via a catheter, is carried by bulk flow though the interstitial space. This method has proved to be a useful laboratory technique for targeted, wide-spread distribution of a broad range of agents including small molecules and gene therapy vectors, such as viruses and liposomes. The success in the lab has translated into a significant number of clinical studies which combine the use of convection-enhanced delivery and elegantly devised molecular strategies. Additionally, several studies suggest that convection-enhanced delivery can be accurately predicted with mathematical models, allowing precise planning of dosage and distribution in the clinical setting. Taken together, the results of these investigations hint at the promise of an expanded armantarium against brain tumors.
Microinjection of the excitatory amino acid glutamate is commonly used to stimulate neuronal cell bodies in brainstem nuclei that are crucial for cardiovascular regulation, respiratory control, and other functions. One such nucleus, the rostral ventrolateral medulla (RVLM), integrates afferent information to coordinate cardiovascular responses to changes in the environment. In the RVLM, an increase in mean blood pressure of ≥25 mmHg following glutamate microinjection is widely accepted as evidence of accurate localisation of this nucleus. However, the dose of glutamate, and injection volume, varies considerably between investigators, and the optimal parameters are controversial. Here we examined data from 34 publications over the past 20 years in which glutamate doses, ranging from 0.0051 to 8.5 μg, were injected to identify the rostral ventrolateral medulla (RVLM) in rat. The aim of this chapter is to describe, in a pragmatic way, the ideal parameters for this method. Our meta-analysis reveals that there is a dose–response relationship between glutamate and blood pressure at low doses, but once the dose is sufficient to elicit a ³35 mmHg rise in blood pressure, the response plateaus (∼0.5 μg). Interestingly, the injection volumes used in the studies examined do not show any correlation with the observed blood pressure responses. Neither strain nor weight of rat had any influence on the blood pressure response induced by glutamate. Glutamate microinjection is a stable and reproducible method for activating cell bodies, but not fibres of passage, in most brain regions, and may also be a useful tool for normalising other drug responses.
A general theory of creep flow is proposed which indicates that the permeability tensor can be represented in terms of only four scalar constants.
Voltammetric electrodes fabricated from carbon fibers have been employed as sensors of fluctuations in the concentration of easily oxidized species in the brains of anesthetized rats. The factors which affect access of these compounds to the electrode surface have been examined. While these electrodes show virtually time-independent chronoamperometric currents in vitro during a 2.5-s potential step, the current shows an increased time dependence when the electrode is implanted in the brain, suggesting an altered diffusion profile obtains in vivo. Values of brain diffusion coefficients for ascorbic acid, dihydroxyphenylacetic acid, and α-methyldopamine were estimated by measuring the time for these species to diffuse to an electrode through brain tissue from a very small injection volume (100 nl). Diffusion coefficients in the brain as measured by this method are found to be approximately one third of the value in pH 7.4 buffer. These data suggest that chronoamperometric currents during long potential steps in vivo involve two types of diffusion: at very short times (approximately 100 ms), molecules are electrolyzed which diffuse to the electrode through a pool of extracellular fluid at the tip of the electrode in which diffusion coefficients are close to those measured in aqueous solution. With longer potential step times, the effects of the reduced diffusion coefficient of these substances through brain tissue is apparent. The utility of these concepts is demonstrated by the rapid response time of the electrode to concentration changes induced by electrical stimulation of dopamine-containing neurons.
The relationship between pressure, ejection duration and volume ejected was experimentally determined in vitro for micropipettes with different external tip diameters. The relationship between ejection duration and ejected volume is linear in the steady state (i.e. with ejection durations of 1 s or longer) and at sufficiently high pressures (above about 100 kPa) and for pipettes with a sufficiently high hydrodynamic conductance (larger than 1 pl s−1 kPa−1 at 230 kPa). In this range, flows were found with low Reynolds numbers (smaller than 10), which is consistent with laminar flows. For all but the largest micropipettes, the relationship between pressure and ejected volume is alinear: the pipettes' apparent hydrodynamic conductances increase with increasing pressure. Micropipettes with apparent hydrodynamic conductances between 0.04 and 1400 pl s−1 kPa−1 (at 230 kPa) were tested. Duration-pressure combinations could be defined where the duration-volume relationship was either linear or monotonic. Such duration-pressure combinations were different for pipettes with different apparent hydrodynamic conductances. A quick method is described to measure the pipette's apparent hydrodynamic conductance at the pressure used, corrected for the fluid's viscosity. Measurement of this conductance permits predictable injections of known volumes of fluid in the range of 100 pl to 1 μl with a precision of 10–20%.
Proofs of the theorems of local volume averaging which relate averages of derivatives to derivatives of averages are presented. A distribution function whose derivatives are proportional to the Dirac delta function is used in the development and the proofs demonstrated are intended to be simpler than those found in the literature.
The amounts of substances delivered by applying pressure to fine. Theta-type, fluid-filled glass microelectrodes were quantitated by in vitro measurement. Relationships between the volumes ejected, the duration of pressure application, and tip size were investigated. The tip sizes ranged between ⩽ 0.6 and 1.2 μm o.d. and were suitable for recording intracellularly from cortical neurons in awake cats.With electrodes of tip sizes ⩾ 0.6 μm, the volumes of fluid ejected increased linearly with time. Volumes were determined by measuring the activity of ejected radio isotopes or the size of droplets of an aqueous solution expressed into mineral oil. Measurements were obtained over periods of up to 180 sec of pressure application (80 psi). The volumes expressed ranged between 101−105 μm3. The rate of ejection increased as the tip size increased. Tips of 0.6 μm passed up to 2 × 102 μm3; 0.9 μm, up to 4 × 103 μm3 and 1.2 μm up to 5 × 104 μm3 volumes for 30 sec of pressure application. The output rate was greater for ejections of ethanolic solution than for aqueous solution.The repeatability of ejection from individual electrodes was also tested. Relatively consistent, reproducible delivery was found for electrodes with tips between 0.6 and 1.2 μm. The results suggest that the pressure microinjection method affords more predictable, quantitative delivery than the iontophoretic method. Femtomolar (10−15 M) amounts of pharmacologically active substances can be administered by such means.
Diffusion coefficients of catecholamine neurotransmitters, their metabolites and related species was measured in brain extracellular fluid using in vivo voltammetric techniques. Nanoliter volumes of the species were pressure-ejected into the rat caudate nucleus and their concentration profiles were determined at nearby voltammetric detector electrodes. Thorough testing was carried out to show that the present methodology gave results which agreed with brain diffusion coefficients measured previously by ion-selective microelectrode techniques. All of the species which are anionic at pH 7.4 have brain diffusion coefficients about one-third of their solution counterparts in accord with earlier studies of diffusion in tortuous media. However, the brain diffusion coefficients of all the cation species are about three-times slower than those of the anions. This phenomenon is believed to be caused by ion binding with the polyanionic glycosaminoglycans and related species in brain tissue. In vitro model experiments lend support to this interpretation. This new information on biogenic amines and their metabolites provides meaningful predictions of the spatio-temporal concentration distribution of these species in the extracellular fluid.
The release of substance P (SP) and two analogues by iontophoresis or pressure from microelectrodes was compared. Substance P was released linearly by iontophoresis from electrodes while no release of the analogues was detected. [N-methylphenylalanine8, N-methylglycine9-] SP5-11 (DiMeC7) and [methyl-2-aminoethyl]11 SP (SP-DAE) were released from electrodes by pressure ejection with linear relationships in all cases between pressure and the amounts released. Under the tested experimental conditions, release of substance P by iontophoresis was between 2 and 3 orders of magnitude less than that by pressure over a given time. The release of substance P and the uncharged analogue DiMeC7 by pressure was very similar while release of SP-DAE was one order of magnitude less.
Alpha-bungarotoxin has been used extensively to characterize the acetylcholine receptor at the myoneural junction and in the electric organ, where it irreversibly blocks the response to acetylcholine. α-Bungarotoxin has also been employed in numerous biochemical studies of presumed acetylcholine receptors in the brain, although physiological studies demonstrating its effectiveness in blocking the response to acetylcholine are lacking, and α-bungarotoxin is ineffective in blocking the response to acetylcholine in sympathetic ganglia neurons. In-this study α-bungarotoxin was applied to the optic tectum of the goldfish and of the marine toad in dilute concentration (10−7m) either directly to the pial surface, or to the tectal neuropil by a micropipette. The latter method of application resulted in the rapid irreversible abolition of excitatory postsynaptic potentials generated in dendrites of tectal neurons by optic nerve activation, as shown by analysis of the laminar current source density. Responses to nonretinal (thalamic) inputs were unaffected, indicating specificity of action. Topical application of α-bungarotoxin was ineffective in the goldfish, and of considerably reduced effectiveness in the toad, due to a substantial diffusion barrier posed by the pia-arachnoid. A mathematical method based on the solution of the differential equation describing simultaneous diffusion and chemical reaction was developed to determine both the diffusion coefficient and the association rate constant for α-bungarotoxin from physiological measurements. We estimate the diffusion coefficient and the Stokes radius of α-bungarotoxin to be approx1.7 × 10−6 cm2/s and 12A˚respectively, and the association rate constant to be 5.1 × 104m−1s−1, in reasonable agreement with values obtained by biochemical methods.
1. The validity of the macroscopic laws of ion diffusion was critically examined within the microenvironment of the extracellular space in the rat cerebellum using ion-selective micropipettes and ionophoretic point sources. 2. The concepts of volume averaging, volume fraction (alpha) and tortuosity (lambda) were defined and shown to be theoretically appropriate for quantifying diffusion in a complex medium such as the brain. 3. Diffusion studies were made with the cations tetramethylammonium and tetraethylammonium and the anions alpha-naphthalene sulphonate and hexafluoro-arsenate, all of which remained essentially extracellular during the measurements. Diffusion parameters were measured for a period of 50s and over distances of the order of 0.1 mm. 4. Measurements of the diffusion coefficients of the ions in agar gel gave values that were very close to those derivable from the literature, thus confirming the validity of the method. 5. Measurements in the cerebellum did not reveal any systematic influences of ionophoretic current strength, electrode separation, anisotropy, inhomogeneity, charge discrimination or uptake, within the limits tested. 6. The pooled data from measurements with all the ions gave alpha = 0.21 +/- 0.02 (mean +/- S.E. of mean) and lambda = 1.55 +/- 0.05 (mean +/- S.E. of mean). 7. These results show that the extracellular space occupies about 20% of the rat cerebellum and that the diffusion coefficient for small monovalent extracellular ions is reduced by a factor of 2.4 (i.e. lambda 2) without regard to charge sign. The over-all effect of this is to increase the apparent strength of any ionic source in the cerebellum by a factor of lambda 2/alpha, about 12-fold in the present case, and to modify the time course of diffusion. 8. These conclusions confirm that the laws of macroscopic diffusion are closely obeyed in the cerebellum for small ions in the extracellular space, provided that volume fraction and tortuosity are explicitly taken into account. It is likely that these conclusions are generally applicable to other brain regions and other diffusing substances.
The micropressure ejection technique was characterized in vivo as well as in vitro. Volumes of radiolabelled drug released from multibarreled pipettes by this method increased linearly with increasing ejection pressure or time. This was reflected in vivo by graded inhibitions of cortical cell activity with increasing ejection pressures of enkephalin. The linearity of this response was independent of interejection time, suggesting that there is no warm-up phenomenon with the micropressure ejection technique. In vitro, interejection times did not affect the amount of 3H-sucrose released by micropressure ejection. The volume of drug released by micropressure ejection was highly reproducible. Likewise, the responses of cerebellar Purkinje cells to repeated pressure applications of norepinephrine or GABA were uniform over long periods of time. The variability in drug release between two barrels of a multibarreled pipette, or between two pipettes using micropressure ejection, was also considerably less than that observed with microiontophoresis. In addition to the linearity and reproducibility of micropressure ejection, these studies demonstrate that substances which are difficult to apply by iontophoresis, such as peptides, are readily applied by this method.