Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice.

Several disorders have been associated with mutations in Na,K-ATPase alpha isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimer's disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the alpha1 isoform is found in many cell types, the alpha2 isoform is predominantly expressed in astrocytes, and the alpha3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the alpha2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the alpha3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous alpha1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the alpha2 and alpha3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders.

Physiological characteristics of the projection pathway from the medial preoptic to the nucleus raphe magnus of the rat and its modulation by the periaqueductal gray.

Anatomical studies have shown a strong projection from the medial preoptic nucleus of the hypothalamus (MPO) to both the periaqueductal gray (PAG) and nucleus raphe magnus (NRM). In this study, we examined the physiological characteristics of MPO to NRM connections and examined how blockade of neuronal transmission and of the glutamatergic system within the PAG modifies this pathway. In deeply anesthetized rats, recordings were made from NRM neurons that were identified by their response to peripheral mechanical stimulation and designated as "E", "I", or "N" if they were excited, inhibited, or not activated by noxious stimulation. In addition, cells were identified as spinally projecting if they could be antidromically activated by stimulation of the dorsolateral funiculus at the thoracic level. The responses of 204 NRM neurons to electrical and 87 cells to both chemical and electrical stimulation of MPO were recorded. The response of NRM neurons to MPO stimulation was highly dependent on the sensory class of these cells. Chemical stimulation of MPO inhibited 50% (16/32) and excited 16% (5/32) of the I-cells. In contrast, 23% (9/39) of the E-cells were inhibited and 49% (19/39) were excited by chemical stimulation of MPO. Electrical stimulation at intensities below 80 microA at 100Hz had similar effects on the two classes of cells; 62% (24/39) of the E-cells and 31% (10/32) of the I cells were excited, and 31% (12/39) of the E-cells and 59% (19/32) of the I-cells were inhibited. The excitatory response to chemical stimulation lasted for an average of 136.8+/-73.2s and inhibitory response lasted for an average of 143.8+/-102.1s. Electrical stimulation of MPO at 1Hz excited 27%, inhibited 3%, and had no effect on 70% of NRM cells. The mean latency to peak excitation was 9.6+/-6.6ms. Antidromic activation of MPO neurons by NRM stimulation showed an average latency of 6.3+/-3.4ms. Blocking the glutamatergic transmission within the PAG (by injecting kynurenic acid (KYN) into the PAG) blocked the inhibitory response of 40% (6/15) of the I-cells and inhibitory response of 43% (3/7) of the E-cells. The excitatory response of 27% (3/11) of the I-cells and the excitatory response of 14% (1/7) of the E-cells were blocked by kynurenic injection into the PAG. It is concluded that: (1) in response to chemical stimulation of MPO, the number of I-cells that were inhibited was more than three times the number of I-cells that were excited; in contrast, the number of E-cells that were excited was more than twice the number of E-cells that were inhibited. (2) The interaction between MPO and NRM can be modulated by blockade of the neuronal transmission or blockade of the glutamatergic system in the PAG. (3) Simultaneous activity of many synapses is required for activation of the MPO-NRM pathway. (4) MPO to NRM interaction is mediated by fibers with a conduction velocity of less than 1m/s.

Partial sciatic nerve ligation results in an enlargement of the receptive field and enhancement of the response of dorsal horn neurons to noxious stimulation by an adenosine agonist.

In this study we examined the effect of partial sciatic nerve ligation (PSNL) on the receptive field size, the baseline firing rate (BFR) and the response of spinal dorsal horn (DH) neurons to mechanical stimulation. In addition, we tested the effect of adenosine agonist, 5'-N-ethylcarboxamide-adenosine (NECA), and the adenosine antagonist caffeine on these parameters. Adult male Sprague-Dawley animals were used. One-third to one-half of the right sciatic nerve was tightly ligated. Unanesthetized animals were tested for their response to mechanical stimulation using Von Frey filaments and a blunt probe. The mean force that produced a paw withdrawal response in the operated animals was significantly less than the force that produced withdrawal in unoperated animals (median: 103.5 vs. 259.7; P < 0.001 for the paw ipsilateral to the ligation). Extracellular recordings were made from nociceptive-specific DH neurons located in laminal I-V of chloral hydrate-anesthetized rats. Recordings were made from 38 neurons in the right and 29 cells in the left DH of unoperated and 40 cells in right and 41 cell in the left DH of operated animals. The BFRs of neurons recorded in the operated animals were not significantly different from those recorded in normal animals. The mean receptive field size (RFS) of neurons (both ipsilateral and contralateral to the ligation) in the operated animals was significantly larger than the RFS of unoperated animals (right side: 180 +/- 2.8 mm2 compared to 66 +/- 2.3 mm2; left side: 93 +/- 31 compared to 65 +/- 21). Twenty-four percent of all neurons in the operated group had bilateral receptive fields; in contrast, only 3% of the neurons in the control animals showed bilateral receptive fields. To examine the effects of adenosine agonist and antagonist, NECA and caffeine were applied next to the recording electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of norepinephrine in the interaction between the lateral reticular nucleus and the nucleus raphe magnus: an electrophysiological and behavioral study.

We have previously demonstrated that the nucleus raphe magnus (NRM) sends a predominantly inhibitory projection to the lateral reticular nucleus (LRN); however, the pharmacology of this pathway is not known. The purpose of this study was to examine the role of norepinephrine in the NRM-LRN system using both electrophysiological and behavioral techniques. Sixty-nine LRN cells were recorded extracellularly. Cells were tested for their response to noxious and innocuous peripheral stimulation applied to the dorsal body surface. The majority of cells were classified as wide dynamic range, with inhibition being the predominant response; receptive fields were located primarily on the tail and hind limbs. The effect of excitatory amino acid glutamate (GLU) administration into NRM (GLU-NRM) was tested on all 69 cells. GLU-NRM inhibited 55 of 69 LRN cells tested; 7 cells were excited and 7 cells did not respond. Thirty-nine LRN cells were tested for their response to norepinephrine (NE) iontophoretically applied in LRN (NE-LRN). Two distinct types of effects were noted. In 9 cells, both NE-LRN and GLU-NRM produced a strong inhibition, with the magnitude of effect between the 2 drugs significantly correlated. In a second group of cells (n = 12), GLU-NRM produced an inhibitory effect while NE-LRN had no effect on the cells' baseline firing rate. However, when the 2 drugs were applied simultaneously, NE-LRN blocked the inhibitory effects of NRM stimulation. The effect of the alpha 2-receptor antagonist yohimbine (YOH) on NRM-evoked responses was tested in 30 LRN cells. The majority of these cells were inhibited by GLU-NRM. Similar to the dichotomous effect noted by NE-LRN, YOH applied iontophoretically in LRN (YOH-LRN) had two predominant effects on NRM-produced inhibition. In 14 of 27 cells, YOH-LRN significantly potentiated the inhibitory effects of NRM stimulation by increasing the duration of the inhibitory epoch an average of 100 sec. In 7 of 27 cells, YOH directly applied in LRN partially antagonized NRM-evoked inhibition. In a second series of experiments, microinjection cannulas were placed within NRM and LRN in order to determine the effect of blocking alpha 2-receptor activity within LRN on NRM stimulation-produced analgesia in an intact animal. Administration of D,L-homocysteic acid in NRM resulted in a significant increase in baseline tail-flick latency of approximately 140%. Pretreatment with YOH (3 micrograms in 0.5 microliter) in LRN resulted in a significant potentiation of this analgesic effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat.

Injection of GABA into the midbrain periaqueductal gray (PAG) activates medullary neurons that are involved in pain inhibition and potentiates morphine-induced analgesia. These observations suggest that GABAergic mechanisms in the PAG may modulate the descending pain inhibitory system that arises from this structure. In the present study, the effects of GABA and GABA antagonists on membrane properties and baseline activity of PAG neurons were examined using both in vitro and in vivo preparations. Application of bicuculline methiodide (BICM), at a dose that blocked the response to GABA, potently increased the baseline firing rate in 53% of cells recorded in vitro and 74% of cells recorded in the intact preparation. Application of BICM often yielded multiple or burst spiking episodes in both preparations. In 69% of cells the effect of BICM was diminished or totally abolished when the slice was perfused with high-magnesium, calcium-free, physiological saline solution. Intracellular recordings revealed that bicuculline caused depolarization of the membrane (70% of cells), increased the firing frequency (94% of cells) and increased the frequency of excitatory postsynaptic potentials (18% of cells). The effect of bicuculline on membrane resistance was not pronounced and in 64% of neurons it did not cause any measurable change in the resting membrane resistance. PAG neurons responsive to GABA and its antagonists were observed in all regions of the PAG. However, the highest number of neurons that responded to GABA and its antgonists was found in the medial and medioventral parts of the PAG. These results indicate that PAG may contain a tonically active GABAergic network that operates, at least in part, through GABAA receptors. This GABAergic system may modulate activity in descending pain inhibitory pathways emanating from PAG.

Membrane and synaptic effects of corticotropin-releasing factor on periaqueductal gray neurons of the rat.

Corticotropin-releasing factor (CRF) has been identified as a major component of the hypothalamic-pituitary-adrenal (HPA) axis. By stimulating the release of adrenocorticotropin hormone (ACTH), CRF acts as a key mediator of the stress response. However, CRF receptors and neuronal elements are present in many extrahypothalamic regions of the brain. A region that contains both CRF-ergic neurons and CRF receptors is the midbrain periaqueductal gray (PAG). The physiological effects of CRF in the PAG are unknown. In this study, an in vitro preparation, extracellular and intracellular patch-clamp recordings, were used to examine the effects of CRF, applied through an injecting electrode, on PAG neurons. Recordings were made from 147 neurons in the PAG. CRF injecting electrode concentrations of 0.05 and 1 microM were tested. At the higher concentration, CRF had a predominant excitatory effect on the neurons, and at the lower concentration, CRF produced no significant effect on the neurons. The excitatory effect was dose dependent and was often associated with a depolarization in membrane potential in intracellular recordings. Application of the CRF antagonist, alpha-helical CRF, blocked this excitatory effect. It is concluded that CRF has a predominant excitatory effect on PAG neurons. It is also concluded that CRF is not acting presynaptically. This excitatory effect of CRF on PAG neurons may lead to activation of a descending analgesic pathway.

Subthalamic nucleus stimulation increases brain derived neurotrophic factor in the nigrostriatal system and primary motor cortex.

The mechanisms underlying the effects of long-term deep brain stimulation of the subthalamic nucleus (STN DBS) as a therapy for Parkinson's disease (PD) remain poorly understood. The present study examined whether functionally effective, long-term STN DBS modulates glial cell line-derived neurotrophic factor (GDNF) and/or brain-derived neurotrophic factor (BDNF) in both unlesioned and unilateral 6-hydroxydopamine lesioned rats. Lesioned rats that received two weeks of continuous unilateral STN DBS exhibited significant improvements in parkinsonian motor behaviors in tests of forelimb akinesia and rearing activity. Unilateral STN DBS did not increase GDNF in the nigrostriatal system, primary motor cortex (M1), or hippocampus of unlesioned rats. In contrast, unilateral STN DBS increased BDNF protein 2-3 fold bilaterally in the nigrostriatal system with the location (substantia nigra vs. striatum) dependent upon lesion status. Further, BDNF protein was bilaterally increased in M1 cortex by as much as 2 fold regardless of lesion status. STN DBS did not impact cortical regions that receive less input from the STN. STN DBS also was associated with bilateral increases in BDNF mRNA in the substantia nigra (SN) and internal globus pallidus (GPi). The increase observed in GPi was completely blocked by pretreatment with 5-Methyl-10,11-dihydro-5 H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), suggesting that the activation of N-methyl-D-aspartate (NMDA) receptors was involved in this phenomenon. The upregulation of BDNF associated with long term STN DBS suggest that this therapy may exert pronounced and underappreciated effects on plasticity in the basal ganglia circuitry that may play a role in the symptomatic effects of this therapy as well as support the neuroprotective effect of stimulation documented in this rat model.

Sensitization of the trigeminal sensory system during different stages of the rat estrous cycle: implications for menstrual migraine.

OBJECTIVES: To determine if the sensitization of the trigeminal system changes after dural activation of the trigeminal nerve during different stages of the rat estrous cycle. BACKGROUND: The specific mechanisms through which ovarian hormones trigger menstrual migraine are currently unknown. Past animal studies have suggested that the response properties of the trigeminal nucleus caudalis (TNC) may change during the different phases of the rat estrous cycle, but none have been performed in an experimental paradigm for migraine headache. METHODS: Sixty-one cycling female Sprague-Dawley rats were used for these experiments. The stage of the estrous cycle of each animal was identified by examination of the cellular morphology of vaginal lavage. The animals were anesthetized and a 7 mm portion of the skull was removed that was centered over the sagittal sinus. A tungsten electrode was used to record from neurons in the TNC or CI-CIII regions. Only neurons that had both dural and cutaneous receptive fields were used for these experiments. Facial receptive field sizes (RFS) were mapped and neurophysiologic response properties of the TNC/CI-CIII neurons to cutaneous and dural stimuli was ascertained before and after application of capsaicin to the dura. One-way and repeated measure analysis of variance were used to compare changes in RFS and response properties of TNC/CI-CIII neurons from animals during different stages of the rat estrous cycle. RESULTS: When data were analyzed individually for each stage, there was greater enlargement of cutaneous receptive fields and enhanced sensitivity of the trigeminal system to cutaneous stimuli during proestrus as compared to metestrus and diestrus after dural activation with capsaicin (P values <.05). When data were pooled from stages with similar hormonal milieus, the percent change in the response magnitude of TNC neurons to electrical stimulation of the dura was greater and receptive field enlargement was larger from the proestrous/estrous group compared to those from the metestrous/diestrous group after administration of capsaicin (P values <.05). CONCLUSIONS: There is enhanced sensitization of the trigeminal system during the later halves of proestrus and estrus, which represent stages of the rat estrous cycle during and immediately following an abrupt decline in ovarian hormones. If similar changes occur during the human menstrual cycle these results could have important implications for menstrual migraine.

The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm.

This study uses genetically altered mice to examine the contribution of the Na(+)-K(+)-ATPase alpha2 catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The alpha2 protein is reduced by 38% in alpha2-heterozygous and absent in alpha2-knockout mice, and alpha1-isoform is upregulated 1.9-fold in alpha2-knockout. Resting potentials are depolarized by 0.8-4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced alpha2 content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the alpha2-isoform at a step after membrane excitation, which cannot be restored simply by increasing alpha1 content. Na+/Ca2+ exchanger expression decreases in parallel with alpha2-isoform, suggesting that Ca2+ extrusion is affected by the altered alpha2 genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban, or plasma membrane Ca(2+)-ATPase. These results demonstrate that the Na(+)-K(+)-ATPase alpha1-isoform alone is able to maintain equilibrium K+ and Na+ gradients and to substitute for alpha2-isoform in most cellular functions related to excitability and force. They further indicate that the alpha2-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction.