Isolated Brainstem Preparation Research Articles

Synaptic Mechanisms of Delay Eyeblink Classical Conditioning: AMPAR Trafficking and Gene Regulation in an In Vitro Model.

An in vitro model of delay eyeblink classical conditioning was developed to investigate synaptic plasticity mechanisms underlying acquisition of associative learning. This was achieved by replacing real stimuli, such as an airpuff and tone, with patterned stimulation of the cranial nerves using an isolated brainstem preparation from turtle. Here, our primary findings regarding cellular and molecular mechanisms for learning acquisition using this unique approach are reviewed. The neural correlate of the in vitro eyeblink response is a replica of the actual behavior, and features of conditioned responses (CRs) resemble those observed in behavioral studies. Importantly, it was shown that acquisition of CRs did not require the intact cerebellum, but the appropriate timing did. Studies of synaptic mechanisms indicate that conditioning involves two stages of AMPA receptor (AMPAR) trafficking. Initially, GluA1-containing AMPARs are targeted to synapses followed later by replacement by GluA4 subunits that support CR expression. This two-stage process is regulated by specific signal transduction cascades involving PKA and PKC and is guided by distinct protein chaperones. The expression of the brain-derived neurotrophic factor (BDNF) protein is central to AMPAR trafficking and conditioning. BDNF gene expression is regulated by coordinated epigenetic mechanisms involving DNA methylation/demethylation and chromatin modifications that control access of promoters to transcription factors. Finally, a hypothesis is proposed that learning genes like BDNF are poised by dual chromatin features that allow rapid activation or repression in response to environmental stimuli. These in vitro studies have advanced our understanding of the cellular and molecular mechanisms that underlie associative learning.

Persistent augmentation of fictive air breathing by hypoxia: An in vitro study of the role of GABAB signaling in pre-metamorphic tadpoles

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations.Addition of baclofen to the aCSF (0,2 μM – 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

Developmental Maturation of Functional Coupling Between Ventilatory Oscillators in the American Bullfrog.

Many vital motor behaviors - including locomotion, swallowing, and breathing - appear to be dependent upon the activity of and coordination between multiple endogenously rhythmogenic nuclei, or neural oscillators. Much as the functional development of sensory circuits is shaped during maturation, we hypothesized that coordination of oscillators involved in motor control may likewise be maturation-dependent, i.e., coupling and coordination between oscillators change over development. We tested this hypothesis using the bullfrog isolated brainstem preparation to study the metamorphic transition of ventilatory motor patterns from early rhythmic buccal (water) ventilation in the tadpole to the mature pattern of rhythmic buccal and lung (air) ventilation in the adult. Spatially distinct oscillators drive buccal and lung bursts in the isolated brainstem; we found these oscillators to be active but functionally uncoupled in the tadpole. Over the course of metamorphosis, the rhythms produced by the buccal and lung oscillators become increasingly tightly coordinated. These changes parallel the progression of structural and behavioral changes in the animal, with adult levels of coupling arising by the metamorphic stage (forelimb eruption). These findings suggest that oscillator coupling undergoes a maturation process similar to the refinement of sensory circuits over development.

Environmentally induced return to juvenile-like chemosensitivity in the respiratory control system of adult bullfrog, Lithobates catesbeianus.

The degree to which developmental programmes or environmental signals determine physiological phenotypes remains a major question in physiology. Vertebrates change environments during development, confounding interpretation of the degree to which development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) produces phenotypes. Tadpoles mainly breathe water for gas exchange and frogs may breathe water or air depending on their environment and are, therefore, exemplary models to differentiate the degree to which life-stage vs. environmental context drives developmental phenotypes associated with neural control of lung breathing. Using isolated brainstem preparations and patch clamp electrophysiology, we demonstrate that adult bullfrogs acclimatized to water-breathing conditions do not exhibit CO2 and O2 chemosensitivity of lung breathing, similar to water-breathing tadpoles. Our results establish that phenotypes associated with developmental stage may arise from plasticity per se and suggest that a developmental trajectory coinciding with environmental change obscures origins of stage-dependent physiological phenotypes by masking plasticity. An unanswered question in developmental physiology is to what extent does the environment vs. a genetic programme produce phenotypes? Developing animals inhabit different environments and switch from one to another. Thus a developmental time course overlapping with environmental change confounds interpretations as to whether development (i.e. permanent processes) or phenotypic plasticity (i.e. reversible processes) generates phenotypes. Tadpoles of the American bullfrog, Lithobates catesbeianus, breathe water at early life-stages and minimally use lungs for gas exchange. As adults, bullfrogs rely on lungs for gas exchange, but spend months per year in ice-covered ponds without lung breathing. Aquatic submergence, therefore, removes environmental pressures requiring lung breathing and enables separation of adulthood from environmental factors associated with adulthood that necessitate control of lung ventilation. To test the hypothesis that postmetamorphic respiratory control phenotypes arise through permanent developmental changes vs. reversible environmental signals, we measured respiratory-related nerve discharge in isolated brainstem preparations and action potential firing from CO2 -sensitive neurons in bullfrogs acclimatized to semi-terrestrial (air-breathing) and aquatic-overwintering (no air-breathing) habitats. We found that aquatic overwintering significantly reduced neuroventilatory responses to CO2 and O2 involved in lung breathing. Strikingly, this gas sensitivity profile reflects that of water-breathing tadpoles. We further demonstrated that aquatic overwintering reduced CO2 -induced firing responses of chemosensitive neurons. In contrast, respiratory rhythm generating processes remained adult-like after submergence. Our results establish that phenotypes associated with life-stage can arise from phenotypic plasticity per se. This provides evidence that developmental time courses coinciding with environmental changes obscure interpretations regarding origins of stage-dependent physiological phenotypes by masking plasticity.

Excitatory and inhibitory synaptic mechanisms at the first stage of integration in the electroreception system of the shark

High impulse rate in afferent nerves is a common feature in many sensory systems that serve to accommodate a wide dynamic range. However, the first stage of integration should be endowed with specific properties that enable efficient handling of the incoming information. In elasmobranches, the afferent nerve originating from the ampullae of Lorenzini targets specific neurons located at the Dorsal Octavolateral Nucleus (DON), the first stage of integration in the electroreception system. Using intracellular recordings in an isolated brainstem preparation from the shark we analyze the properties of this afferent pathway. We found that stimulating the afferent nerve activates a mixture of excitatory and inhibitory synapses mediated by AMPA-like and GABAA receptors, respectively. The excitatory synapses that are extremely efficient in activating the postsynaptic neurons display unusual voltage dependence, enabling them to operate as a current source. The inhibitory input is powerful enough to completely eliminate the excitatory action of the afferent nerve but is ineffective regarding other excitatory inputs. These observations can be explained by the location and efficiency of the synapses. We conclude that the afferent nerve provides powerful and reliable excitatory input as well as a feed-forward inhibitory input, which is partially presynaptic in origin. These results question the cellular location within the DON where cancelation of expected incoming signals occurs.

Localization of essential rhombomeres for respiratory rhythm generation in bullfrog tadpoles using a binary search algorithm: Rhombomere 7 is essential for the gill rhythm and suppresses lung bursts before metamorphosis

Frog metamorphosis includes transition from water breathing to air breathing but the extent to which such a momentous change in behavior requires fundamental changes in the organization of the brainstem respiratory circuit is unknown. Here, we combine a vertically mounted isolated brainstem preparation, "the Sheep Dip," with a search algorithm used in computer science, to identify essential rhombomeres for generation of ventilatory motor bursts in metamorphosing bullfrog tadpoles. Our data suggest that rhombomere 7, which in mammals hosts the PreBötC (PreBötzinger Complex; the likely inspiratory oscillator), is essential for gill and buccal bursts. Whereas rhombomere 5, in close proximity to a brainstem region associated with the mammalian expiratory oscillator, is essential for lung bursts at both stages. Therefore, we conclude there is no rhombomeric translocation of respiratory oscillators in bullfrogs as previously suggested. In premetamorphic tadpoles, functional ablation of rhombomere 7 caused ectopic expression of precocious lung bursts, suggesting the gill oscillator suppresses an otherwise functional lung oscillator in early development.

Transmission of the respiratory rhythm to trigeminal and hypoglossal motor neurons in the American Bullfrog (Lithobates catesbeiana)

Spatially distinct, interacting oscillators in the bullfrog medulla generate and coordinated buccal and lung ventilatory rhythms, but how these rhythms are transmitted onto trigeminal and hypoglossal motor neurons is unknown. Using a vertically-mounted isolated brainstem preparation, the Sheep Dip, we identified the regions of the brainstem containing motor nuclei using a solution capable of blocking synaptic release and, following washout, locally exposed these regions to 5μM NBQX and/or 50μM AP5. Local application of NBQX significantly reduced the amplitude of buccal and lung bursts on the trigeminal nerve, and lung bursts on the hypoglossal nerve. Local AP5 caused a significant reduction in lung burst amplitude on both nerves, but for buccal bursts, hypoglossal amplitude increased and trigeminal amplitude was unchanged. Local co-application of NBQX and AP5 eliminated fictive respiratory motor output completely in both nerves. These results are consistent with mammalian data, suggesting a critical role for glutamate in transmission of respiratory activity from oscillators to motor neurons.

Neuronal Mechanisms of Respiratory Pattern Generation Are Evolutionary Conserved

A brainstem region, the paratrigeminal respiratory group (pTRG), has been suggested to play a crucial role in the respiratory rhythm generation in lampreys. However, a detailed characterization of the pTRG region is lacking. The present study performed on isolated brainstem preparations of adult lampreys provides a more precise localization of the pTRG region with regard to both connectivity and neurochemical markers. pTRG neurons projecting to the vagal motoneuronal pool were identified in a restricted area of the rostral rhombencephalon at the level of the isthmic Müller cell I1 close to sulcus limitans of His. Unilateral microinjections of lidocaine, muscimol, or glutamate antagonists into the pTRG inhibited completely the bilateral respiratory activity. In contrast, microinjections of glutamate agonists enhanced the respiratory activity, suggesting that this region is critical for the respiratory pattern generation. The retrogradely labeled pTRG neurons are glutamatergic and surrounded by terminals with intense substance P immunoreactivity. Cholinergic neurons were seen close to, and intermingled with, pTRG neurons. In addition, α-bungarotoxin binding sites (indicating nicotinic receptors) were found throughout the pTRG area and particularly on the soma of these neurons. During apnea, induced by blockade of ionotropic glutamate receptors within the same region, microinjections of 1 μm substance P or 1 mm nicotine into the pTRG restored rhythmic respiratory activity. The results emphasize the close similarities between the pTRG and the mammalian pre-Bötzinger complex as a crucial site for respiratory rhythmogenesis. We conclude that some basic features of the excitatory neurons proposed to generate respiratory rhythms are conserved throughout evolution.

Rhythmic jaw movement in the isolated brainstem preparation from neonatal mice

Rhythmic jaw movement in the isolated brainstem preparation from neonatal mice

Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition ofIn VitroEyeblink Classical Conditioning

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Respiratory rhythms generated in the lamprey rhombencephalon

Brainstem networks generating the respiratory rhythm in lampreys are still not fully characterized. In this study, we described the patterns of respiratory activities and we identified the general location of underlying neural networks. In a semi-intact preparation including the brain and gills, rhythmic discharges were recorded bilaterally with surface electrodes placed over the vagal motoneurons. The main respiratory output driving rhythmic gill movements consisted of short bursts (40.9±15.6 ms) of discharge occurring at a frequency of 1.0±0.3 Hz. This fast pattern was interrupted by long bursts (506.3±174.6 ms) recurring with an average period of 37.4±24.9 s. After isolating the brainstem by cutting all cranial nerves, the frequency of the short respiratory bursts did not change significantly, but the slow pattern was less frequent. Local injections of a glutamate agonist (AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or d,l-amino-5-phosphonopentanoic acid (AP5)) were made over different brainstem regions to influence respiratory output. The results were similar in the semi-intact and isolated-brainstem preparations. Unilateral injection of AP5 or CNQX over a rostral rhombencephalic region, lateral to the rostral pole of the trigeminal motor nucleus, decreased the frequency of the fast respiratory rhythm bilaterally or stopped it altogether. Injection of AMPA at the same site increased the rate of the fast respiratory rhythm and decreased the frequency of the slow pattern. The activity recorded in this area was synchronous with that recorded over the vagal motoneurons. After a complete transverse lesion of the brainstem caudal to the trigeminal motor nucleus, the fast rhythm was confined to the rostral area, while only the slow activity persisted in the vagal motoneurons. Our results support the hypothesis that normal breathing depends on the activity of neurons located in the rostral rhombencephalon in lampreys, whereas the caudal rhombencephalon generates the slow pattern.

Lung and Buccal Ventilation in the Frog: Uncoupling Coupled Oscillators

The frog, with two distinct ventilatory acts, provides a useful model to investigate the prospective interaction of two oscillators in generating the respiratory rhythm. Building on evidence supporting the existence of separate oscillators generating buccal and lung ventilation, we have attempted to uncouple the two rhythms in the isolated brain stem preparation. Opioid preferentially inhibits the lung rhythm, suggesting an uncoupling of the lung from the buccal oscillator. Reduction of the superfusate chloride concentration alters both the buccal and the lung rhythms. Joint application of opioid and reduced-chloride superfusate leads to an increase in the variability of the buccal burst-to-lung burst intervals. This increase in variability suggests that chloride-mediated mechanisms are involved in coupling the buccal oscillator to the lung oscillator. Given the results from these interventions, we propose a simple schematic model of the frog respiratory rhythm generator, outlining the coupling of the lung and buccal oscillators.

Nitric oxide changes its role as a modulator of respiratory motor activity during development in the bullfrog ( Rana catesbeiana)

Nitric oxide (NO) is a unique chemical messenger that has been shown to play a role in the modulation of breathing in amphibians and other vertebrates. In the post-metamorphic tadpole and adult amphibian brainstem, NO, acting via the neuronal isoform of nitric oxide synthase (nNOS), is excitatory to the generation of lung burst activity. In this study, we examine the modulation of breathing by NO during development of the amphibian brainstem. Isolated brainstem preparations from pre-metamorphic and late-stage post-metamorphic tadpoles ( Rana catesbeiana) were used to determine the role of NO in modulating central respiratory neural activity. Respiratory neural activity was monitored with suction electrodes recording extracellular activity of cranial nerve rootlets that innervate respiratory musculature. Brainstems were superfused with an artificial cerebrospinal fluid (aCSF) at 20–22 °C containing l-nitroarginine ( l-NA; 1–10 mM), a non-selective NOS inhibitor. In pre-metamorphic tadpoles, l-NA increased fictive gill ventilation frequency and amplitude, and increased lung burst frequency. By contrast, l-NA applied to the post-metamorphic tadpole brainstem had little effect on fictive buccal activity, but significantly decreased lung burst frequency and the frequency of lung burst episodes. These data indicate that early in development, NO provides a tonic inhibitory input to gill and lung burst activity, but as development progresses, NO provides an excitatory input to lung ventilation. This changing role for NO coincides with the shift in importance in the different respiratory modes during development in amphibians; that is, pre-metamorphic tadpoles rely predominantly on gill ventilation whereas post-metamorphic tadpoles have lost the gills and are obligate air-breathers primarily using lungs for gas exchange. We hypothesize that NO provides a tonic input to the respiratory CPG during development and this changing role reflects the modulatory influence of NO on inhibitory or excitatory modulators or neurotransmitters involved in the generation of respiratory rhythm.

Development of the respiratory response to hypoxia in the isolated brainstem of the bullfrogRana catesbeiana

The aim of this study was to examine the effects of cellular hypoxia, and the contribution of anaerobic metabolism, on respiratory activity in bullfrogs at different stages of development. Respiratory-related neural activity was recorded from cranial nerve rootlets in isolated brainstem preparations from pre-metamorphic (Taylor-Kollros (T-K) stages VIII-XVI) and postmetamorphic tadpoles (T-K stages XXIV-XXV) and adults. Changes in fictive gill/lung activity in brainstems from pre-metamorphic tadpoles and lung activity in postmetamorphic tadpoles and adults were examined during superfusion with control (98% O(2)/2% CO(2)) or hypoxic (98% N(2)/2% CO(2)) artificial cerebrospinal fluid (aCSF). Iodoacetate (IAA; 100 micromol l(-1)) was used in conjunction with hypoxic aCSF to inhibit glycolysis. Gill burst frequency in pre-metamorphic brainstems did not change over a 3 h exposure to hypoxia and fictive lung burst frequency slowed significantly, but only after 3 h hypoxia. Blockade of glycolysis with IAA during hypoxia significantly reduced the time respiratory activity could be maintained in pre-metamorphic, but not in adult, brainstems. In brainstems from post-metamorphic tadpoles and adults, lung burst frequency became significantly more episodic within 5-15 min hypoxic exposure, but respiratory neural activity was subsequently abolished in every preparation. The cessation of fictive breathing was restored to control levels upon reoxygenation. Neither tadpole nor adult brainstems exhibited changes in neural bursts resembling 'gasping' that is observed in mammalian brainstems exposed to severe hypoxia. There was also a significant increase in the frequency of 'non-respiratory' bursts in hypoxic postmetamorphic and adult brainstems, but not in pre-metamorphic brainstems. These results indicate that pre-metamorphic tadpoles are capable of maintaining respiratory activity for 3 h or more during severe hypoxia and rely to a great extent upon anaerobic metabolism to maintain respiratory motor output. Upon metamorphosis, however, hypoxia results in significant changes in respiratory frequency and pattern, including increased lung burst episodes, non-ventilatory bursts and a reversible cessation of respiratory activity. Adults have little or no ability to maintain respiratory activity through glycolysis but, instead, stop respiratory activity until oxygen is available. This 'switch' in the respiratory response to hypoxia coincides morphologically with the loss of gills and obligate air-breathing in the postmetamorphic frog. We hypothesize that the cessation of respiratory activity in post-metamorphic tadpoles and adults is an adaptive, energy-saving response to low oxygen.

Respiratory survival mechanisms in acetylcholinesterase knockout mouse

Cholinergic neurotransmission ensures muscle contraction and plays a role in the regulation of respiratory pattern in the brainstem. Inactivation of acetylcholinesterase (AChE) by organophosphates produces respiratory failure but AChE knockout mice survive to adulthood. Respiratory adaptation mechanisms which ensure survival of these mice were examined in vivo by whole body plethysmography and in vitro in the neonatal isolated brainstem preparation. AChE-/- mice presented no AChE activity but unaffected butyrylcholinesterase (BChE) activity. In vivo, bambuterol (50-500 microg/kg s.c.) decreased BChE activity peripherally but not in brain tissue and induced apnea and death in adult and neonate AChE-/- mice without affecting littermate AChE+/+ and +/- animals. In vitro, bath-applied bambuterol (1-100 microm) and tetraisopropylpyrophosphoramide (10-100 microm) decreased BChE activity in the brainstem but did not perturb central respiratory activity recorded from spinal nerve rootlets. In vitro, the cholinergic agonists muscarine (50-100 microm) and nicotine (0.5-10 microm) induced tonic activity in respiratory motoneurons and increased the frequency of inspiratory bursts in AChE+/+ and +/- animals. These effects were greatly attenuated in AChE-/- animals. The results suggest that, in mice lacking AChE, (i) BChE becomes essential for survival peripherally but plays no critical role in central rhythm-generating structures and (ii) a major adaptive mechanism for respiratory survival is the down-regulated response of central respiratory-related neurons and motoneurons to muscarinic and nicotinic agonists.

Effect of serotonin (5-HT) on trigeminal rhythmic activities generated in in vitro brainstem block preparations.

We used rat isolated brainstem block preparations to analyze the functional roles of serotonin receptors in the generation of trigeminal rhythmic activities. We previously reported that trigeminal rhythmic activities could be induced by some pharmacological applications in an isolated brainstem preparation with a rostral boundary at the border between the inferior and superior colliculus, and a caudal border at the level of the rostral facial nucleus. However, the same stimulation did not induce trigeminal rhythmic activities in a whole brainstem block preparation with the same rostral boundary and a caudal border at the obex level. In the present study, both the 5-HT(1A) phthalimido-butyl-piperazine, and the 5-HT(2C) agonist, 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane, combined with N-methyl-D,L-aspartate and bicuculline, elicited trigeminal rhythmic activities in a whole brainstem block preparation. Our results suggest that serotonin has both facilitation and inhibition effects on the generation of trigeminal rhythmic activities in an isolated brainstem block preparation in vitro.

Properties of LTD and LTP of retinocollicular synaptic transmission in the developing rat superior colliculus.

The developing retinocollicular pathway undergoes synaptic refinement in order to form the precise retinotopic pattern seen in adults. To study the mechanisms which underlie refinement, we investigated long-term changes in retinocollicular transmission in rats aged P0-P25. Field potentials (FPs) in the superior colliculus (SC) were evoked by stimulation of optic tract fibers in an in vitro isolated brainstem preparation. High intensity stimulation induced long-term depression (LTD) in the SC after both low (1000 stimuli at 1 Hz) and higher (1000 stimuli at 50 Hz) frequency stimulation. The induction of LTD was independent of activation of NMDA and GABA(A) receptors, because D-APV (100 microM) and bicuculline (10 microM) did not block LTD. Induction of LTD was dependent upon activation of L-type Ca(2+) channels as 10 microM nitrendipine, an L-type Ca(2+) channel blocker, significantly decreased the magnitude of LTD. LTD was down-regulated during development. LTD magnitude was greatest in rats aged P0-P9 and significantly less in rats aged P10-P25. Long-term potentiation (LTP) was induced by low intensity stimulation and only after high frequency tetanus (1000 stimuli at 50 Hz). LTP was NMDA receptor dependent because d-APV (100 microM) completely abolished it. LTP induction was also blocked by the L-type Ca2+ channel blocker nitrendipine. The magnitude of LTP first increased with age, being significantly greater at P7-P13 than at P0-3 and then decreased at P23-25. In summary, both LTD and LTP are present during retinocollicular pathway refinement, but have different transmitter and ionic mechanisms and time courses of expression.

Temperature and pH/CO 2 modulate respiratory activity in the isolated brainstem of the bullfrog ( Rana catesbeiana)

The effects of temperature and pH/CO 2 were examined in isolated brainstem preparations from adult North American bullfrogs ( Rana catesbeiana). These experiments were undertaken to determine the effects of temperature on fictive breathing, central pH/CO 2 chemoreception, and to examine potential alphastat regulation of respiration in vitro. Adult bullfrog brainstem preparations were isolated, superfused with an artificial cerebrospinal fluid (aCSF) and respiratory-related neural activity was recorded from cranial nerves V, X and XII. In Series I experiments ( N=8), brainstem preparations were superfused with aCSF equilibrated with 2% CO 2 at temperatures ranging from 10 to 30 °C. Neural activity was present in all preparations in the temperature range of 15–25 °C, but was absent in most preparations when aCSF was at 10 or 30 °C. The absence of fictive breathing at high (30 °C) temperatures was transient since fictive breathing could be restored upon returning the preparation to 20 °C. In Series II experiments ( N=10), preparations were superfused with aCSF equilibrated with 0%, 2% and 5% CO 2 at temperatures of 15, 20 and 25 °C. Fictive breathing frequency ( f R) was significantly dependent upon aCSF pH at all three temperatures, with slopes ranging from −0.82 min −1 pH unit −1 (15 °C) to −3.3 min −1 pH unit −1 (20 °C). There was a significant difference in these slopes ( P<0.02), indicating that central chemoreceptor sensitivity increased over this temperature range. Fictive breathing frequency was significantly dependent upon the calculated alpha-imidazole (α Im) ionization ( P<0.05), consistent with the alphastat hypothesis for the effects of temperature on the regulation of ventilation. However, most of the variation in f R was not explained by α Im ( R 2=0.05), suggesting that other factors account for the regulation of fictive breathing in this preparation. The results indicate that the in vitro brainstem preparation of adult bullfrogs has a limited temperature range (15–25 °C) over which fictive breathing is consistently active. Although there is a close correspondence of ventilation in vitro and in vivo at low temperatures, these data suggest that, as temperature increases, changes in ventilation in the intact animal are likely to be more dependent upon peripheral feedback which assumes a greater integrative role with respect to chemoreceptor drive, respiratory frequency and tidal volume.

Evolution of central respiratory chemoreception: a new twist on an old story

Evolution of central respiratory chemosensitivity has been linked traditionally to the need for carbon dioxide regulation that accompanied the evolution of air breathing in terresterial animals. We examined the validity of this linkage by investigating the possibility of central chemoreception in air breathing fish that diverged from the amphibian lineage long before the appearance of terrestriality. We showed that the isolated brainstem preparation of the long nose gar ( Lepisosteus osseus) produces a putative motor pattern for lung ventilation, which is responsive to CO 2. These findings, together with more inferential evidence, suggest an association between air breathing and central chemosensitivity in aquatic animals that spans the major branches in vertebrate phylogeny. Furthermore, developmental observations in tadpoles suggest that the neural substrates for central chemoreception exist in proximity to that for rhythm generation. We postulate that a primitive ancestral CPG, sensitive to CO 2 is conserved and is evidenced in the intrinsic coupling of respiratory CPG and central chemoreception in modern tetrapods.

Synaptic Regulation of L-Type Ca2+Channel Activity and Long-Term Depression during Refinement of the Retinocollicular Pathway in Developing Rodent Superior Colliculus

The retinocollicular pathway undergoes activity-dependent refinement during postnatal development, which results in the precise retinotopic order seen in adults. This process is NMDA- and nitric oxide-dependent. Recent studies have shown that L-type Ca2+ channels may also play a role in synaptic plasticity, but such channel activity has not previously been reported in the developing superior colliculus (SC). Here we report the presence of a postsynaptic plateau potential mediated by L-type Ca2+ channels using whole-cell current clamp of the SC in an isolated brainstem preparation of rats. Seventy percent of SC neurons showed these potentials as early as postnatal day 0 (P0)-P2. The potential was blocked by nitrendipine and/or APV and facilitated by bicuculline, showing that the channel is activated by NMDA receptor-mediated EPSPs and deactivated by GABAA receptor-mediated IPSPs. Blockade of L-type Ca2+ channels also diminished long-term depression, which we could induce in the retinocollicular pathway in neonatal animals. The incidence of plateau potentials decreased to 39% of neurons by P10-P14, suggesting that L-type calcium channels may contribute to retinocollicular pathway refinement in the developing SC.