Chronic Animal Preparations Research Articles

Large-Scale Fluorescence Calcium-Imaging Methods for Studies of Long-Term Memory in Behaving Mammals.

During long-term memory formation, cellular and molecular processes reshape how individual neurons respond to specific patterns of synaptic input. It remains poorly understood how such changes impact information processing across networks of mammalian neurons. To observe how networks encode, store, and retrieve information, neuroscientists must track the dynamics of large ensembles of individual cells in behaving animals, over timescales commensurate with long-term memory. Fluorescence Ca(2+)-imaging techniques can monitor hundreds of neurons in behaving mice, opening exciting avenues for studies of learning and memory at the network level. Genetically encoded Ca(2+) indicators allow neurons to be targeted by genetic type or connectivity. Chronic animal preparations permit repeated imaging of neural Ca(2+) dynamics over multiple weeks. Together, these capabilities should enable unprecedented analyses of how ensemble neural codes evolve throughout memory processing and provide new insights into how memories are organized in the brain.

Localization of Phonation Integration Center in Rabbits

Problem The midbrain periaqueductal gray (PAG) serves as a phonation integration center, coordinating respiratory, laryngeal, and orofacial activity during vocalization. The objective of this pilot study was to explore electrical excitation of the midbrain PAG for eliciting vocalization using an in-vivo rabbit preparation and determine the feasibility of using image guidance for improved localization of target midbrain structures. Methods Pilot experiments were conducted using four New Zealand White (NZW) breeder rabbits. In the first experiment, a stereotaxic frame was used to guide stimulation of the midbrain PAG without the use of image guidance. Decisions regarding track placements were directed by a stereotaxic atlas of the NZW rabbit brain. Histologic stains of axial brain sections were then obtained to determine electrode track positioning. In a subsequent experiment, magnetic resonance imaging (MRI) and computed tomography (CT) scans were obtained and used to localize midbrain structures for advanced planning capabilities. Results Without the use of image guidance, phonation was feasible, measuring 80–100 Hz using electrical excitation of the midbrain PAG (0.5 to 1.5 mA, 200 Hz, 2 second train duration). However, precise localization of the midbrain PAG proved technically challenging. Using CT and MRI data, scans could be loaded onto an image guided surgical system and combined using an image fusion tool for advanced planning. Conclusion The authors demonstrate the feasibility of electrical excitation of the midbrain PAG to produce vocalization in rabbits using a stereotaxic brain atlas and offer an alternative approach using image guidance for advanced planning and more precise localization of target midbrain structures. Significance The biomechanical forces of phonation and dynamic nature of vocal fold remodeling have created a need for chronic animal preparations to investigate these reparative processes in-vivo. Image guided techniques may improve the feasibility of brain stimulation techniques for eliciting and studying time dependent changes in vocal fold remodeling. Support NIH-NIDCD R03 DC008400.

Complex effects of general anesthesia on sensory processing in the spinal dorsal horn

A chronic animal preparation allowed us to compare activity of the same single, spinal dorsal horn neurons in the physiologically intact, awake, drug-free state and in the anesthetized state. The inhalation anesthetic enflurane produced profound, and at times, opposite effects on spinal dorsal horn neuron responses to non-noxious and noxious receptive field stimulation. Some effects would not have been predicted, based upon current understanding of anesthetics.

In-situ calibration of the implantable force transducer

Recently, an implantable force transducer (IFT) has been introduced [Xu et al. (1992, J. Biomech. Engng 114, 170–177)] which can be used in tight spaces where force recordings with established transducers, such as the buckle-type transducers, are not possible because of impingement artifacts. The IFT is easily implanted in chronic animal preparations; however, calibration of the IFT in terminal experiments has produced unreliable results. The problems of IFT calibration are that minute movements of the transducer within the tendon, slight misalignments of the tendon, or slight errors in the line of pull cause dramatic changes in the IFT voltage output for a given applied calibration load. Here, we propose a method that eliminates the above calibration problems primarily because the target tendon is left in situ, the calibration loads are applied by the muscles which insert into the target tendon, and the transducer is implanted into the target tendon about two weeks prior to calibration. The theoretical and experimental approaches are demonstrated for the cat patellar tendon, but in principle can be performed with any tendon. The results are repeatable, lie within expected values, and reproduce some of the basic properties which have been observed in prior IFT testing.

In-situ calibration of the implantable force transducer

Recently, an implantable force transducer (IFT) has been introduced [Xu et al. (1992, J. Biomech. Engng 114 , 170–177)] which can be used in tight spaces where force recordings with established transducers, such as the buckle-type transducers, are not possible because of impingement artifacts. The IFT is easily implanted in chronic animal preparations; however, calibration of the IFT in terminal experiments has produced unreliable results. The problems of IFT calibration are that minute movements of the transducer within the tendon, slight misalignments of the tendon, or slight errors in the line of pull cause dramatic changes in the IFT voltage output for a given applied calibration load. Here, we propose a method that eliminates the above calibration problems primarily because the target tendon is left in situ , the calibration loads are applied by the muscles which insert into the target tendon, and the transducer is implanted into the target tendon about two weeks prior to calibration. The theoretical and experimental approaches are demonstrated for the cat patellar tendon, but in principle can be performed with any tendon. The results are repeatable, lie within expected values, and reproduce some of the basic properties which have been observed in prior IFT testing.

Synaptic plasticity in the thalamo-cortical pathway as one of the neurobiological correlates of forelimb flexion conditioning: electrophysiological investigation in the cat

1. In a previous study, using a chronic cat preparation subjected to an associative conditioning procedure, we described the plasticity of the thalamo-cortical pathway by qualitatively and quantitatively analyzing the motor responses induced by stimulating each of the relays on the cerebello-thalamo-cortical pathway. In the present study, it was proposed to analyze the effects on the synapses located between thalamic endings and cortical neurones, using a twofold behavioral and electrophysiological approach, with a view to correlating the patterns of synaptic plasticity with the changes in the motor responses recorded. 2. For this purpose, a reduced, functionally organized sensorimotor circuit, which can be taken to be a neuronal analog of associative conditioning, was studied in an awake chronic animal preparation. This circuit was defined on the basis of the sites at which conditioned (CS) and unconditioned stimuli (UCS) were applied: the CS was applied at a site on the cerebellar interpositus nucleus which activated the forepaw musculature so as to induce flexion movements and the UCS was applied to the skin of the distal part of that paw so as to induce reflex flexion movements. By repetitively activating the central nervous pathways by the associated CS and UCS according to a predefined temporal pattern, the efficiency of the thalamo-cortical pathway's contribution to the movement production was enhanced, and its capacity to convey the cerebellar inputs to neurons in the motor cortex increased. 3. The associative nature of the conditioning was tested using previously established criteria. The setting up of motor and central changes in response to the repetitive presentation of paired CS and UCS, the fact that these changes were reversible because they could be abolished by applying extinction procedures, and the consistency of their occurrence whenever the CS was applied repeatedly alone for several days to naive animals, all showed that the stimuli of both kinds (CS and UCS) had to be applied together for the plasticity of the thalamo-cortical pathway to be expressed. 4. By determining whether the waves constituting the cerebello-cortical responses were excitatory or inhibitory, the nature of the changes in the transmission of the cerebellar impulses to neurons in the motor cortex was established.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholecystokinin-induced contraction of opossum sphincter of Oddi

In this study, we evaluated the mechanism of action whereby cholecystokinin increases spike-burst rate of the opossum sphincter of Oddi (SO). Each spike burst corresponds to a peristaltic SO contraction. Two types of animal preparations were evaluated: (1) awake chronic animal preparations and (2) anesthetized animals. A total of 19 chronic animals were prepared by implantation of electrodes on the SO, gastric antrum, duodenum, and jejunum. SO spike-burst rate was stimulated by intravenous infusion of CCK-OP (10 ng/kg/min), feeding, or intraduodenal infusion of fat-containing nutrient. Each stimulus was begun 20 min after cessation of phase III duodenal MMC activity and caused an increase in SO spike-burst rate from about 2 to 6/min that lasted for less than or equal to 1 hr. Such increases were antagonized substantially by hexamethonium, atropine, or methysergide. The CCK antagonist, L364718, antagonized the excitatory SO response to CCK-OP infusion or intraduodenal infusion of fat-containing nutrient (Isocal) but did not antagonize the response to feeding; CR1409 had no antagonistic effect on SO response to any of the three types of stimuli. In the acute studies in anesthetized animals, an intravenous bolus dose of CCK-OP (800 ng/kg) caused a substantial increase in SO spike-burst rate that was antagonized by CR1409 but not by atropine, hexamethonium, methysergide, L364718, or TTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of Cholecystokinin-Induced Contraction of the Opossum Gallbladder

The present study evaluated the mechanism of cholecystokinin-induced gallbladder contraction in awake opossums. Each of 19 chronic animal preparations had an indwelling gallbladder cannula for monitoring changes in gallbladder volume and a jugular catheter for administration of cholecystokinin octapeptide and drugs. An intraduodenal catheter allowed intraduodenal infusion of Isocal (Mead Johnson Laboratories, Evansville, Ind.). Bipolar electrodes in the stomach, duodenum, and jejunum enabled monitoring of the duodenal migratory myoelectric complex cycle. One-hour infusions of cholecystokinin octapeptide (10 ng/kg/min), intraduodenal Isocal (0.4 ml/min), or feeding were started 20 min after cessation of phase 3 duodenal migratory myoelectric complex activity. Bolus intravenous doses of potential pharmacological antagonists were given 10 min before and 20 min after the onset of cholecystokinin octapeptide infusion, Isocal infusion, or feeding. Each test challenge induced about 60% gallbladder emptying at 30 min and 70% at 60 min. Cholecystokinin octapeptide-induced gallbladder emptying was converted to filling by hexamethonium and nearly abolished by atropine. Similar results were obtained for gallbladder emptying induced by feeding or duodenal infusion of Isocal. Phentolamine caused a modest decrease of cholecystokinin octapeptide-induced gallbladder contraction at 30 min, whereas postprandial or Isocal-induced gallbladder contraction was unaffected. Cholecystokinin octapeptide-induced gallbladder contraction was not affected by pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide, prazosin, pyrilamine, cimetidine, methysergide, naloxone, or propranolol. In acute studies of anesthetized animals, gallbladder contraction induced by a D100 intravenous bolus of cholecystokinin octapeptide (800 ng/kg) was not antagonized by hexamethonium, atropine, or tetrodotoxin. It is concluded (a) that cholecystokinin-induced physiologic contraction of the opossum gallbladder occurs through neural mechanisms rather than by a direct action of cholecystokinin on gallbladder smooth muscle, and (b) that studies using pharmacologic bolus doses of cholecystokinin octapeptide and tetrodotoxin indicate that cholecystokinin receptors exist on the gallbladder smooth muscle which do not seem to have any physiological role in gallbladder emptying.

Fetal and Newborn Lambs Differ in Their Cardiopulmonary Responsiveness to Adenosine Agonists

We investigated cardiorespiratory responses to adenosine analogs in the pre- and postnatal periods in an unanesthetized chronic animal preparation. Heart rate, blood pressure, breathing movements and blood gases were measured in 8 fetal lambs (117-130 days gestation) and 9 newborn lambs (4-30 days). Various doses of analogs were given by intravenous infusion. L-N6-5'-Phenylisopropyladenosine (L-PIA), 5-N-ethyl carboxamidoadenosine (NECA) and cyclohexyladenosine (CHA) were studied. All analogs produced dose-dependent bradycardia and hypotension in the fetus. However, in the newborn, NECA produced a dose-dependent tachycardia, whereas PIA and CHA produced a dose-dependent bradycardia. Fetal breathing movements were interrupted by all the analogs, but they did not produce apnea in the newborn. No changes in blood gases were observed. The actions of the adenosine agonists were blocked by caffeine.

Transtracheal stimulation of the recurrent laryngeal nerve

Phonation was induced in 10 mongrel dogs under general anesthesia by way of transtracheal stimulation of the recurrent laryngeal nerves under conditions of constant air flow. Although stimulation voltages were approximately 10 times the voltage required for direct stimulation, no cardiac or respiratory abnormalities were observed. Photoglottographic and electroglottographic signals were the same for both direct and transtracheally induced phonation. This phonation induction method can be used in chronic animal preparations to study vocal fold vibration sequentially and may be clinically applied to the treatment of patients with laryngeal problems that have failed to respond, or are not amenable, to standard forms of vocal rehabilitation.

Laryngeal muscle and diaphragmatic activities n conscious dog pups

A new chronic animal preparation ( i.e. puppies) for the study of respiratory laryngeal muscle acitivity is described. Posterior cricoarytenoid (PCA), thyroarytenoid (TA) and diaphragmatic activities were monitored in conscious puppies (13–43 days old) using chronically implanted electrodes. PCA activity was prominent during inspiration in all behavioral states. TA and post-inspiratory diaphragmatic activities were consistently high during expiration in quiet wakefulness and quiet sleep, but expiratory TA activity decreased dramatically and post-inspiratory diaphragmatic activity became highly variable in rapid eye movement sleep. There was also a tendency for these expiratory muscle activities to become less pronounced with increasing postnatal age. When a chronically implanted, low resistnace T-tube tracheal cannula was suddenly opened, thus effectively eliminating the effects of upper airway resistance on airflow, respiratory frequency dramatically increased. These results indicate that the exhibits muscle activities consistent with the maintenance of an elevated end-expiratory lung volume achieved by the retardation of expiratory airflow using the larynx and diaphragm.

Changes in ovine fetal hindlimb amino acid metabolism during maternal fasting.

The flux of various substrates across the ovine fetal and maternal hindlimbs was measured in the fed state and after 5 days of maternal fasting. Whole blood concentrations of glucose, oxygen, ammonia, and six amino acids (glutamate, glutamine, alanine, valine, isoleucine, and leucine) were determined in the fetal and maternal femoral artery and distal inferior vena cava in 15 chronic animal preparations. During fasting the fetal arterial glucose concentration fell by 40% (from 0.828 to 0.494 mM), and the arteriovenous concentration difference decreased by 30% (from 0.148 to 0.099 mM). Similar changes were noted in maternal blood. Fetal oxygen concentrations remained similar between the fed and fasted state, and the fetal arteriovenous oxygen concentration difference increased slightly from 0.861 to 1.02 mM. The glucose oxygen quotient decreased in the fetus from 1.20 to 0.621. In addition, significant changes occurred in the net balance of several amino acids during the fasted state. Both alanine and glutamine, which demonstrated a positive uptake by the fetal hindlimb during the fed state, showed a substantial efflux from the fetal hindlimb during the fasting period. The fetal arteriovenous concentration difference of the branched-chain amino acids (leucine, isoleucine, and valine) increased significantly during fasting. These observations are consistent with the hypothesis that the ovine fetus adapts to a diminished supply of glucose from the mother by enhanced amino acid catabolism and, possibly, proteolysis with subsequent release of gluconeogenic precursors in the form of alanine and glutamine.

An intracranial electrode delivery system for the chronic mouse and bat preparation

The construction of an electrode delivery device is described which facilitates the accurate placement of multiple electrode arrays in the chronic small animal preparation. The device is easily constructed, easily used, and has many features which can be readily modified by individual investigators to serve their own needs. During implantation of the electrodes, the delivery device can be used to stimulate, lesion or to record the EEG activity from discrete neural sites. The electrode array is permanently affixed to the skull by dental acrylic anchored to two cranial screws. The use of a delivery system such as the one described may encourage neurobehavioral research with the mouse, a more easily maintained and economical subject than the standard laboratory rat.

185 COMPARISON OF UMBILICAL GLUCOSE UPTAKE AND FETAL GLUCOSE TURNOVER RATES

Net fetal glucose uptake has been measured by us previously in fetal lambs using the Fick Principle. This study compares simultaneous measurements of net fetal glucose uptake with fetal glucose turnover rates, using primed-constant infusions of 14C-l-glucose and either 3H-2 or 3H-6-glucose carried out in chronic animal preparations. The glucose turnover rates of placenta plus fetus, expressed as mg min−1 Kg−1 were: 12.02±1.35 SEM (14C label) and 13.78±1.61 SEM (3H label). However, the placenta accounted for a large fraction of these turnover rates. Forty to seventy percent of the infused labeled glucose was taken up by the placenta. The glucose turnover rates of the fetal body (mg min Kg−1) were: 5.22±0.62 SEM (14C label) and 8.85±1.63 SEM (3H label). The simultaneously measured fetal glucose uptake via the umbilical circulation was 4.81±0.37 SEM (mg min−1 Kg−1). In 4 of 6 animals, fetal glucose uptake and the turnover rate of 14C-1-glucose by the fetal body were virtually equal. Of the infused radioactive glucose, ∼10% was converted to CO2 by the fetus and ∼ 7% to lactate by the placenta. Previous studies of fetal glucose turnover have not discriminated between placental uptake and turnover rate within the fetus. This study demonstrates such discrimination is feasible and should lead to a more meaningful comparison between fetal and neonatal metabolism.

Construction of wire leads and electrodes for use in slow wave recording in small animals

Techniques are described for the construction of wire electrodes and leads for use in recording slow wave activity in chronic animal preparations. Advantages of the technique include durability, lightness and absence of significant interference with animal movement.

An experimental test of diaschisis

The diaschisis theory of recovery of function was directly tested for the first time. Using the dentate gyrus in adult rats as the model system, extracellular field potentials elicited by commissural stimulation were monitored in both acute and chronic animal preparations before and from 0.5 hr to 11 days following the lesion. The lesion resulted in the loss of the commissurally elicited long-latency potential but did not disrupt the short-latency monosynaptic potential. No changes in latency, amplitude, form, or stimulus threshold in the monosynaptic potential could be detected.

Doppler Blood Flow Measurements in Chronic Animal Preparations

Doppler Blood Flow Measurements in Chronic Animal Preparations

A device for measuring myocardial contractility

To obtain quantitative information about myocardial contractility in acute and chronic animal preparations during drug administration, a device for computing the ratio dp/dt: integrated isometric tension has been developed. Based on analogue computer techniques, the instrument samples ventricular pressure and the rate of change of ventricular pressure (dp/dt), during that phase of isovolumic contraction occurring before peak dp/dt. The isometric tension obtained is integrated and stored. The peak value of dp/dt is also stored. The two values are then simultaneously sampled, the division is performed by an analogue divider, and a 100 ms pulse whose height is proportional to the ratio (and thus myocardial contractility) is fed to a chart recorder. The R wave of the e.c.g. is used to start sampling of the ventricular pressure wave. A pulse derived from the peak dp/dt terminates sampling, and initiates resetting of the instrument ready for the next R wave. In this way, a beat-to-beat measure of myocardial contractility is obtained.

A-V differences of free fatty acids and glycerol in the ovine umbilical circulation.

Conclusions1. Measurement of venous-arterial differences across the umbilical circulation of the sheep fetus in utero and across the maternal uterine circulation in the chronic animal preparation shows no significant passage of free fatty acids to the fetus. This is true despite relatively high maternal blood free fatty acid concentrations. There is a small venous-arterial difference of glycerol across the umbilical circulation; this is, however, sufficient to account for only a small portion of the fetal oxygen consumption due to substances other than glucose. Free fatty acids and glycerol therefore do not appear to constitute a significant part of the metabolic fuel supplied by the mother to the sheep fetus in utero.2. The fetuses of fasted ewes show an elevation of blood free fatty acid and glycerol concentrations. Measurements of venous-arterial differences across the umbilical circulation indicate that this free fatty acid and glycerol rise is of fetal origin.3. Fetal infusion of norepinephrine resul...

Abolition of a conditioned, surface-negative, cortical potential during cryogenic blockade of the non-specific thalamo-cortical system

Cryoprobes were implanted bilaterally in the region of the inferior thalamic peduncle (ITP) of three chronic cat preparations in order to block the non-specific thalamo-cortical system. Three other chronic animal preparations had cryoprobes placed more laterally in this forebrain region in the medial part of the anterior limb of the internal capsule. All six animals had stimulating electrodes placed in nucleus ventralis lateralis (VL) to produce augmenting responses and in nucleus centralis medialis (NCM) to produce recruiting responses. Transcortical recording electrodes were implanted ipsilaterally in the anterior sigmoid, coronal, anterior lateral and anterior suprasylvian cortices. 1. 1. Stimulation of NCM produced either no sustained steady-potential shift or a small positive one, whereas repetitive stimulation of VL produced a train of augmenting responses superimposed upon a large, negative, steady-potential shift which was followed after the cessation of stimulation by a post-stimulus wave of 300–400 msec duration. 2. 2. Reversible cryogenic blockade in the region of the ITP sufficient to abolish recruiting responses abolished or markedly reduced the VL-elicited steady potential and post-stimulus wave recorded in the anterior sigmoid and coronal cortices but did not affect the simultaneously recorded positive-negative augmenting-response components. Cryogenic blockade in the medial part of the anterior limb of the internal capsule did not affect the VL-elicited steady potential or post-stimulus wave, although it did reduced the amplitude of the augmenting response in the anterior sigmoid cortex. 3. 3. The three animals with cryoprobes in the ITP were exposed to a sensory-sensory conditioning paradigm in which a tone was reinforced with a mild electric shock to the skin of the neck. During early conditioning, surface-negative steady-potentials were recorded in the anterior sigmoid, coronal, and anterior lateral, i.e., marginal, cortices. After 500 additional trials, the surface-negative response had a longer latency of onset, occurring just before the start of the shock, and after overtraining, the conditioned negative response was recorded only in the frontal cortical region (anterior sigmoid). 4. 4. Cryogenic blockade in the region of the ITP abolished or markedly reduced the amplitude of the conditioned negative response recorded in the frontal cortical area during both early conditioning and overtraining. 5. 5. During ITP blockade, the simultaneous effect on the steady-potential shift, post-stimulus wave, and conditioned cortical response suggests a common underlying mechanism. 6. 6. It was shown that the VL-elicited steady-potential shift and post-stimulus wave recorded in the frontal and sensory-motor cortex, as well as the conditioned negative response recorded in the frontal cortical region, are affected by the non-specific thalamo-cortical system, a system that from previous studies has been shown to mediate recruiting responses, regulate specific sensory evoked potentials and underlie the performance of certain complex behavioral tasks. The previous work as well as the present is suggestive of a neural mechanism related to the focusing of attention, expectancy and selective perception.