Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 µM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 µM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 µM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 µM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 µM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

HEALTH ASSESSMENT OF CAPTIVE BEARDED VULTURES (GYPAETUS BARBATUS) IN CATALONIA (SPAIN).

Bearded vulture (Gypaetus barbatus) populations are declining worldwide primarily due to anthropogenic factors. A captive breeding program has been established in Spain, a country with one of the largest free-living populations in Europe, to further enhance the conservation efforts of this emblematic species. However, captive vulture populations can be exposed to different risks through food, such as drug residues and antimicrobial-resistant (AMR) bacteria. Health surveillance of species involved in captive breeding programs is important to face introduction of healthy animals in situ and to obtain baseline clinical data. The objective of this study was to assess the general health status of bearded vultures held in captivity in Catalonia (northeastern Spain) by carrying out hematologic, biochemical, toxicologic, and bacteriologic analyses. A total of 16 bearded vultures were sampled; the data obtained from one vulture, with a chronic tibiotarsal fracture, were excluded from the statistical analysis. Hematologic and biochemical parameters of the bearded vultures were mostly within the range of standard values as stated in previous studies. Basal feather and serum corticosterone levels were analyzed and described for the first time in this species. A total of 15 Escherichia coli isolates were obtained that were resistant to fluoroquinolones (80%), tetracycline (60%), trimethoprim and ampicillin (40%), sulfamethoxazole (33%), and colistin (20%), with 40% of them being multidrug resistant. Three of 15 isolates were carriers of the mcr-1 gene. Only the injured bird previously treated with enrofloxacin was positive for fluoroquinolone residues. Periodic monitoring for the presence of AMR bacteria would be recommended in captive breeding programs as a preventive action to establish future therapies.

Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 µm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 µm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

Molecular Detection and Epidemiology of Potentially Zoonotic Cryptosporidium spp. and Giardia duodenalis in Wild Boar (Sus scrofa) from Eastern Spain.

The protozoans Giardia duodenalis and Cryptosporidium spp. are common causes of gastrointestinal disease in humans and animals. While both are commonly documented in domestic animals, few studies have analysed their presence in wildlife. To assess the prevalence of both parasites in wild boar (Sus scrofa) in the Valencian Community (eastern Spain), 498 wild boar faecal samples were collected from 2018 to 2022. Cryptosporidium spp. was detected by performing a nested PCR targeting a 578 bp sequence of the small subunit ribosomal RNA gene (SSU rRNA), followed by sequencing and phylogenetic analysis. For G. duodenalis, a qPCR amplifying a fragment of 62 bp from the SSU rRNA was employed. Positive samples were genotyped for glutamate dehydrogenase and ß-giardin genes. Different epidemiological factors were considered potential modulating variables in the transmission of both parasites. G. duodenalis prevalence was 1.20%, while Cryptosporidium spp. prevalence reached 21.7%. Coinfection was observed in 0.2%. Genotyping of G. duodenalis isolates only detected genotype E. Two species of Cryptosporidium spp. were identified: Cryptosporidium scrofarum and Cryptosporidium suis. The results of this study demonstrate that the exposure to Cryptosporidium spp. in wild boars is high, particularly among young individuals belonging to the Typical Mediterranean climate. Moreover, the probability of infection is dependent on both the season and the density of wild boars. On the other side, exposure to G. duodenalis seems scarce and is influenced, in turn, by the climate. Both Cryptosporidium species detected in the present study have been reported in humans. Due to wild boar increasing in number and their colonisation of urban and peri-urban areas, this could represent an inherent health risk for the human population.

Impact of exposure to urban air pollution on grey squirrel (Sciurus carolinensis) lung health.

The increased rate of global urbanisation has recently exacerbated the significant public health problem of traffic related air pollution. Despite the known significant impact on human health, little is known about the effects of air pollution on wildlife health. The lung is the primary target organ for the effects of exposure to air pollution, leading to lung inflammation, altering the lung epigenome, culminating in respiratory disease. In this study, we aimed to assess lung health and DNA methylation profiles in Eastern grey squirrel (Sciurus carolinensis) populations living across an urban-rural air pollution gradient. Squirrel lung health was assessed in four populations situated across the most polluted inner-city boroughs to the less polluted edges of Greater London. We also assessed lung DNA methylation across three London sites and a further two rural sites in Sussex and North Wales. Lung and tracheal diseases were present in 28% and 13% of the squirrels respectively. Specifically, focal inflammation (13%), focal macrophages with vacuolated cytoplasm (3%) and endogenous lipid pneumonia (3%). There was no significant difference in prevalence of lung, tracheal diseases, anthracosis (carbon presence) or lung DNA methylation levels between urban sites and urban and rural sites respectively or NO2 levels. BALT (Bronchus-Associated Lymphoid Tissue) was significantly smaller in the site with highest NO2 and contained the highest carbon loading compared to sites with lower NO2, however differences in carbon loading in between sites were not significant. High pollution site individuals also had significantly higher numbers of alveolar macrophages which suggests that grey squirrels are exposed to and respond to traffic-related air pollution and further research is needed to understand the impact of traffic-related air pollutants on wildlife health.

Glutamate modulates the firing rate in oculomotor nucleus motoneurons as a function of the recruitment threshold current.

Studies in alert preparations have demonstrated that ocular motoneurons exhibit a phasic­tonic firing rate related to eye velocity and position, respectively. The slopes of these relationships are higher in motoneurons with higher recruitment threshold and have been proposed to depend upon synaptic input. To investigate this hypothesis, motoneurons of the rat oculomotor nucleus were recorded in a brain slice preparation in control conditions and during glutamate (5 µm) application to the bath. Glutamate did not affect membrane potential or input resistance, but produced a decrease in rheobase and depolarization voltage as a function of the current needed for generating a maintained repetitive discharge (recruitment threshold current). In addition, glutamate compressed the range of recruitment threshold current (0.1­0.4 nA) as compared to the control (0.15­0.7 nA). Glutamate exposed motoneurons showed an increase in the tonic frequency gain and the peak frequency. Such increments depended on the recruitment threshold current and the last recruited motoneurons almost doubled the tonic frequency gain (35.2 vs. 57.9 spikes s(−1) nA(−1)) and the peak frequency (52.4 vs. 102.6 spikes s(−1)). Finally, glutamate increased the spike frequency adaptation due to a significant increase in the phasic firing component as compared to the tonic one. In conclusion, glutamate modulates tonic and phasic discharge properties as a function of the recruitment threshold current and, presumably, motoneuron size. These findings contribute to understand the link between cellular functions and motoneuron discharge during oculomotor behaviour.

Clearance and persistence of Escherichia coli in the freshwater mussel Unio mancus.

The excessive use of antibiotics has led to the emergence of resistant bacteria, mainly from the Enterobacterales group, with high pathogenic/zoonotic potentials that can lead to problems in public health. The increasing presence in freshwater ecosystems highlights the need to evaluate potential sentinel species as risk indicators for both ecosystem and human health. The freshwater mussels provide several ecosystem services, may represent potential sentinel species due to their ability to filter water and retain both organic and inorganic particles. We tested the capability of U. mancus to retain Escherichia coli as a model bacterial organism. Under experimental conditions, the mussels could clear suspended E. coli, facilitating its rapid elimination from water within the first 24 h after exposure. The species also presented a maximum retention time of 4 days. We also provide allometric equations correlating the filtering capacity with the length and the weight of mussel body parts often used in biometric studies. We provide a first assessment of the potential of the bivalve Unio mancus to act as a sentinel species for the detection of Enterobacterales and demonstrate the ability to act as a water cleaner.

Antimicrobial Resistance in Pasteurellaceae Isolates from Pyrenean Chamois (Rupicapra pyrenaica) and Domestic Sheep in an Alpine Ecosystem.

Antimicrobial resistance (AMR) has spread worldwide due to the inappropriate use of antimicrobial drugs in human and veterinary medicine, becoming a public health problem. However, little is known about its occurrence and maintenance in wild animals, and very few studies have been carried out in ecosystems subjected to low human pressure. In our study, nasal and lung swabs were collected from hunted Pyrenean chamois (Rupicapra pyrenaica), and nasal swabs from sympatric domestic sheep were also collected. The swabs were cultured in agar plates to obtain bacterial isolates from the Pasteurellaceae family. The presence of AMR was assessed in a total of 28 Pasteurellaceae isolates from 45 Pyrenean chamois, and 9 isolates from sympatric domestic sheep found in the National Hunting Reserve of Freser-Setcases (Northeastern Pyrenees, Spain). The isolates belonged to one of the following three species: Pasteurella multocida, Mannheimia haemolytica and Bibersteinia trehalosi. Some P. multocida and M. haemolytica isolates tested positive for AMR. The statistical analysis revealed no differences between the AMR levels from chamois and domestic sheep isolates. However, one P. multocida of chamois origin presented resistance to cephalosporins and fluoroquinolones, which are antibiotics of critical importance for human health. Further studies are required to elucidate potential routes of dissemination of AMR genes in natural environments and assess any significant persistence in wildlife to design risk mitigation actions.

Assessing methods to live-capture wild boars (Sus scrofa) in urban and peri-urban environments.

BACKGROUND: Wild boar (Sus scrofa) populations are increasing worldwide and invading urban areas. Live-capture can improve the management of this challenge, maximising efficiency, allowing scientific studies and potentially improving animal welfare. This study assesses teleanaesthesia, drop-net, corral trap and cage trap to live-capture wild boar in urban and peri-urban areas, evaluating efficiency and animal stress through haematology and serum biochemistry. METHODS: From 2012 to 2018, 655 wild boars were captured in 279 operations (drop-net=17, teleanaesthesia=186, cage trap=66 and corral trap=10) in the urban and peri-urban areas of Barcelona (Spain). Haematological and serum biochemical variables were determined in 145 wild boars (42 drop-netted, 41 teleanaesthetised, 38 cage-trapped and 24 corral-trapped). RESULTS: Performance (wild boars captured per operation) was highest for drop-net, followed by corral and cage traps, and finally teleanaesthesia. The three physical capture methods were more stressful than teleanaesthesia, causing a more intense physiological reaction, muscular damage, renal function impairment and homeostasis adaption. Stress response was predominantly adrenergic for drop-net and cortisol-induced for cage and corral traps. CONCLUSION: Teleanaesthesia is the choice in reactive urban situations thanks to its adaptability; drop-net effectively targets wild boars in peri-urban environments; cage and corral traps are useful as long-term methods in specific areas.

Co-localization of nitric oxide synthase and choline acetyltransferase in the brain of the goldfish (Carassius auratus).

This work investigates the nitrergic and cholinergic systems in the brain and spinal cord of the goldfish (Carassius auratus). We studied the immunohistochemical localization of antibodies against the neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) by bright-field and confocal microscopy. Nitrergic and cholinergic cells were segregated within the telencephalon, in both dorsal and ventral areas, and co-distributed in some nuclei of the diencephalon, mesencephalon, rhombencephalon, and spinal cord. Double-labeling experiments revealed nNOS/ChAT-positive cells in (1) the diencephalon: the preoptic and suprachiasmatic nuclei, the habenula, the dorsal thalamus, and the nucleus of the medial longitudinal fasciculus; (2) the mesencephalon: the optic tectum, the mesencephalic portion of the trigeminal nucleus, the oculomotor and trochlear nuclei, and the Edinger-Westphal nucleus; and (3) the rhombencephalon: the secondary gustatory nucleus, the nucleus isthmi, the lateral lemniscus nucleus, the cerebellum, the reticular formation, different nuclei of the octaval column, the motor zone of the vagal lobe, and the trigeminal, facial, abducens, glosso-pharyngeal, vagal, and hypobranchial motor nuclei. Double-labeled cells were also observed in the spinal motor column. The percentage of double-labeled cells was different in each studied nucleus, indicating a selective distribution pattern. Because double-labeled cells were more abundant in those nuclei involved with sensory and motor physiological processes, we suggest the involvement of both nitric oxide and acetylcholine in these neural functions in fish.

Preparado anatómico de la cara visceral del hígado humano para evidenciar las ramas de la vena porta hepática aplicando las técnicas de repleción, disección y conservación Laskowski / Anatomical preparation of the visceral aspect of the human liver to demonstrate the branches of the hepatic portal vein applying the Laskowski filling, dissection and conservation techniques

RESUMEN El curso de anatomía humana es crucial en la formación de los estudiantes de medicina y, debido a ello, la escasez de preparados anatómicos significa una dificultad en la enseñanza. El presente estudio tiene como objetivo realizar un preparado anatómico de la cara visceral del hígado que permita visualizar las ramas de la vena porta hepática mediante la aplicación de las técnicas de repleción, disección y conservación Laskowski. En el preparado, se logró visualizar la distribución de las venas suprahepáticas y las ramas de la vena porta que irrigan sus respectivos segmentos, sin encontrar variantes anatómicas. Mediante este trabajo, se logró crear un preparado anatómico que permite visualizar principalmente las ramas de la vena porta hepática. Se recomienda realizar una inspección de la pieza anatómica antes de realizar el trabajo y realizar pruebas para determinar la cantidad adecuada de tinte vegetal a utilizar.

ABSTRACT The human anatomy course is crucial in the education of medical students, and therefore, the shortage of anatomical specimens poses a challenge in teaching. This study aims to create an anatomical preparation of the visceral surface of the liver, allowing visualization of the branches of the hepatic portal vein using the repletion, dissection, and Laskowski preservation techniques. The preparation successfully revealed the distribution of suprahepatic veins and branches of the portal vein irrigating their respective segments, without encountering anatomical variations. Through this study, successfully, produced an anatomical specimen primarily showcasing the branches of the hepatic portal vein. It is recommended inspecting the anatomical specimen before commencing the work, as well as conducting tests to determine the appropriate amount of plant dye to use.

Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus).

The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.

Connections of eye-saccade-related areas within mesencephalic reticular formation with the optic tectum in goldfish.

Physiological studies demonstrate that separate sites within the mesencephalic reticular formation (MRF) can evoke eye saccades with different preferred directions. Furthermore, anatomical research suggests that a tectoreticulotectal circuit organized in accordance with the tectal eye movement map is present. However, whether the reticulotectal projection shifts with the gaze map present in the MRF is unknown. We explored this question in goldfish, by injecting biotin dextran amine within MRF sites that evoked upward, downward, oblique, and horizontal eye saccades. Then, we analyzed the labeling in the optic tectum. The main findings can be summarized as follows. 1) The MRF and the optic tectum were connected by separate axons of the tectobulbar tract. 2) The MRF was reciprocally connected mainly with the ipsilateral tectal lobe, but also with the contralateral one. 3) The MRF received projections chiefly from neurons located within intermediate and deep tectal layers. In addition, the MRF projections terminated primarily within the intermediate tectal layer. 4) The distribution of labeled neurons in the tectum shifted with the different MRF sites in a manner consistent with the tectal motor map. The area containing these cells was targeted by a high-density reticulotectal projection. In addition to this high-density topographic projection, there was a low-density one spread throughout the tectum. 5) Occasionally, boutons were observed adjacent to tectal labeled neurons. We conclude that the organization of the reticulotectal circuit is consistent with the functional topography of the MRF and that the MRF participates in a tectoreticulotectal feedback circuit.

Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS) is caused by a failure of neuronal cells to express the gene encoding the fragile mental retardation protein (FMRP). Clinical features of the syndrome include intellectual disability, learning impairment, hyperactivity, seizures and anxiety. Fmr1 knockout (KO) mice do not express FMRP and, as a result, reproduce some FXS behavioral abnormalities. While intrinsic and synaptic properties of excitatory cells in various part of the brain have been studied in Fmr1 KO mice, a thorough analysis of action potential characteristics and input-output function of CA1 pyramidal cells in this model is lacking. With a view to determining the effects of the absence of FMRP on cell excitability, we studied rheobase, action potential duration, firing frequency-current intensity relationship and action potential after-hyperpolarization (AHP) in CA1 pyramidal cells of the hippocampus of wild type (WT) and Fmr1 KO male mice. Brain slices were prepared from 8- to 12-week-old mice and the electrophysiological properties of cells recorded. Cells from both groups had similar resting membrane potentials. In the absence of FMRP expression, cells had a significantly higher input resistance, while voltage threshold and depolarization voltage were similar in WT and Fmr1 KO cell groups. No changes were observed in rheobase. The action potential duration was longer in the Fmr1 KO cell group, and the action potential firing frequency evoked by current steps of the same intensity was higher. Moreover, the gain (slope) of the relationship between firing frequency and injected current was 1.25-fold higher in the Fmr1 KO cell group. Finally, AHP amplitude was significantly reduced in the Fmr1 KO cell group. According to these data, FMRP absence increases excitability in hippocampal CA1 pyramidal cells.

Avoidance response in goldfish: emotional and temporal involvement of medial and lateral telencephalic pallium.

The hippocampus and the amygdala are involved in avoidance learning in mammals. The medial and lateral pallia of actinopterygian fish have been proposed as homologous to the mammalian pallial amygdala and hippocampus, respectively, on the basis of neuroanatomical findings. This work was aimed at studying the effects of ablation of the medial telencephalic pallia (MP) and lateral telencephalic pallia (LP) in goldfish on the retention of a conditioned avoidance response previously acquired in two experimental conditions. In the first experiment, fish were trained in nontrace avoidance conditioning. In the second experiment, fish were trained in trace avoidance conditioning in which temporal cues were crucial for the learning process. An MP lesion affected the retention of the avoidance response in both procedures; in contrast, an LP lesion impaired the retention only in the trace-conditioning procedure. These data support the presence of two different systems of memory in fish, based on discrete telencephalic areas: the MP, involved in an emotional memory system; and the LP, involved in a spatial, relational, or temporal memory system. Moreover, these differential effects were similar to those produced by amygdalar and hippocampal lesions in mammals. We conclude that these specialized systems of memory could have appeared early during phylogenesis and could have been conserved throughout vertebrate evolution.

Lesions of the medial pallium, but not of the lateral pallium, disrupt spaced-trial avoidance learning in goldfish (Carassius auratus).

The effects of telencephalic lesions of the medial pallium (MP) and lateral pallium (LP) of goldfish on avoidance learning were studied in a two-way, shuttle response, spaced-trial avoidance conditioning situation. Animals received one trial per day, a training regime that permits the assessment of avoidance learning in the absence of stimulus carry-over effects from prior trials. Control and LP-lesioned goldfish exhibited significantly faster avoidance learning than MP-lesioned animals. These results suggest that the MP, but not the LP, is responsible for the widely described deficits in avoidance learning after lesions of the entire telencephalon. The proposal of a functional similarity between the fish MP and the mammalian amygdala, known to be involved in fear conditioning, suggests a conservative phylogenetic role of this area in avoidance learning.

Diminution of voltage threshold plays a key role in determining recruitment of oculomotor nucleus motoneurons during postnatal development.

The size principle dictates the orderly recruitment of motoneurons (Mns). This principle assumes that Mns of different sizes have a similar voltage threshold, cell size being the crucial property in determining neuronal recruitment. Thus, smaller neurons have higher membrane resistance and require a lower depolarizing current to reach spike threshold. However, the cell size contribution to recruitment in Mns during postnatal development remains unknown. To investigate this subject, rat oculomotor nucleus Mns were intracellularly labeled and their electrophysiological properties recorded in a brain slice preparation. Mns were divided into 2 age groups: neonatal (1-7 postnatal days, n = 14) and adult (20-30 postnatal days, n = 10). The increase in size of Mns led to a decrease in input resistance with a strong linear relationship in both age groups. A well-fitted inverse correlation was also found between input resistance and rheobase in both age groups. However, input resistance versus rheobase did not correlate when data from neonatal and adult Mns were combined in a single group. This lack of correlation is due to the fact that decrease in input resistance of developing Mns did not lead to an increase in rheobase. Indeed, a diminution in rheobase was found, and it was accompanied by an unexpected decrease in voltage threshold. Additionally, the decrease in rheobase co-varied with decrease in voltage threshold in developing Mns. These data support that the size principle governs the recruitment order in neonatal Mns and is maintained in adult Mns of the oculomotor nucleus; but during postnatal development the crucial property in determining recruitment order in these Mns was not the modifications of cell size-input resistance but of voltage threshold.

GABAergic Projections to the Oculomotor Nucleus in the Goldfish (carassius Auratus).

The mammalian oculomotor nucleus receives a strong γ-aminobutyric acid (GABA)ergic synaptic input, whereas such projections have rarely been reported in fish. In order to determine whether this synaptic organization is preserved across vertebrates, we investigated the GABAergic projections to the oculomotor nucleus in the goldfish by combining retrograde transport of biotin dextran amine, injected into the antidromically identified oculomotor nucleus, and GABA immunohistochemistry. The main source of GABAergic afferents to the oculomotor nucleus was the ipsilateral anterior octaval nucleus, with only a few, if any, GABAergic neurons being located in the contralateral tangential and descending nuclei of the octaval column. In mammals there is a nearly GABAergic inhibitory inputs; thus, the vestibulooculomotor GABAergic circuitry follows a plan that appears to be shared throughout the vertebrate phylogeny. The second major source of GABAergic projections was the rhombencephalic reticular formation, primarily from the medial area but, to a lesser extent, from the inferior area. A few GABAergic oculomotor projecting neurons were also observed in the ipsilateral nucleus of the medial longitudinal fasciculus. The GABAergic projections from neurons located in both the reticular formation surrounding the abducens nucleus and the nucleus of the medial reticular formation have primarily been related to the control of saccadic eye movements. Finally, all retrogradely labeled internuclear neurons of the abducens nucleus, and neurons in the cerebellum (close to the caudal lobe), were negative for GABA. These data suggest that the vestibuloocular and saccadic inhibitory GABAergic systems appear early in vertebrate phylogeny to modulate the firing properties of the oculomotor nucleus motoneurons.

Changes in somatodendritic morphometry of rat oculomotor nucleus motoneurons during postnatal development.

This work investigates the somatodendritic shaping of rat oculomotor nucleus motoneurons (Mns) during postnatal development. The Mns were functionally identified in slice preparation, intracellularly injected with neurobiotin, and three-dimensionally reconstructed. Most of the Mns (approximately 85%) were multipolar and the rest (approximately 15%) bipolar. Forty multipolar Mns were studied and grouped as follows: 1-5, 6-10, 11-15, and 21-30 postnatal days. Two phases were distinguished during postnatal development (P1-P10 and P11-P30). During the first phase, there was a progressive increase in the dendritic complexity; e.g., the number of terminals per neuron increased from 26.3 (P1-P5) to 47.7 (P6-P10) and membrane somatodendritic area from 11,289.9 microm(2) (P1-P5) to 19,235.8 microm(2) (P6-P10). In addition, a few cases of tracer coupling were observed. During the second phase, dendritic elongation took place; e.g., the maximum dendritic length increased from 486.7 microm (P6-P10) to 729.5 microm in adult Mns, with a simplification of dendritic complexity to values near those for the newborn, and a slow, progressive increase in membrane area from 19,235.8 microm(2) (P6-P10) to 24,700.3 microm(2) (P21-P30), while the somatic area remained constant. In conclusion, the electrophysiological changes reported in these Mns with maturation (Carrascal et al. [2006] Neuroscience 140:1223-1237) cannot be fully explained by morphometric variations; the dendritic elongation and increase in dendritic area are features shared with other pools of Mns, whereas changes in dendritic complexity depend on each population; the first phase paralleled the establishment of vestibular circuitry and the second paralleled eyelid opening.

Muscarinic modulation of recruitment threshold and firing rate in rat oculomotor nucleus motoneurons.

Above recruitment threshold, ocular motoneurons (Mns) show a firing rate linearly related with eye position. Current hypothesis suggests that synaptic inputs are determinant for establishing the recruitment threshold and firing rate gain in these Mns. We investigated this proposal by studying the cholinergic modulation in oculomotor nucleus Mns by intracellular recordings in rat brain slice preparation. All recorded Mns were silent at their resting membrane potential. Bath application of carbachol (10 microm) produced a depolarization and a sustained firing that was not silenced on returning membrane potential to the precarbachol value via DC injection. In response to similar membrane depolarization or equal-current steps, carbachol-exposed Mns produced a higher firing rate and a shorter spike afterhyperpolarization phase with lower amplitude. The relationship between injected current and firing rate (I-F) was linear in control and carbachol-exposed Mns. The slope of these relationships (I-F gain) decreased with carbachol exposure. Bath application of agonist and antagonist of nicotinic and muscarinic acetylcholine receptors in addition to immunohistochemical studies support the notion that muscarinic receptors are primarily involved in the preceding responses. We conclude that muscarinic inputs play an important role in determining the recruitment threshold and firing rate gain observed in oculomotor Mns in vivo.