Nonmuscle myosin IIB as a therapeutic target for the prevention of relapse to methamphetamine use.

Memories associated with drug use increase vulnerability to relapse in substance use disorder (SUD), and there are no pharmacotherapies for the prevention of relapse. Previously, we reported a promising finding that storage of memories associated with methamphetamine (METH), but not memories for fear or food reward, is vulnerable to disruption by actin depolymerization in the basolateral amygdala complex (BLC). However, actin is not a viable therapeutic target because of its numerous functions throughout the body. Here we report the discovery of a viable therapeutic target, nonmuscle myosin IIB (NMIIB), a molecular motor that supports memory by directly driving synaptic actin polymerization. A single intra-BLC treatment with Blebbistatin (Blebb), a small-molecule inhibitor of class II myosin isoforms, including NMIIB, produced a long-lasting disruption of context-induced drug seeking (at least 30 days). Further, postconsolidation genetic knockdown of Myh10, the heavy chain of the most highly expressed NMII in the BLC, was sufficient to produce METH-associated memory loss. Blebb was found to be highly brain penetrant. A single systemic injection of the compound selectively disrupted the storage of METH-associated memory and reversed the accompanying increase in BLC spine density. This effect was specific to METH-associated memory, as it had no effect on an auditory fear memory. The effect was also independent of retrieval, as METH-associated memory was disrupted 24 h after a single systemic injection of Blebb delivered in the home cage. Together, these results argue for the further development of small-molecule inhibitors of NMII as potential therapeutics for the prevention of SUD relapse triggered by drug associations.

Distinct effect of pregnenolone sulfate on NMDA receptor subtypes.

Using rapid agonist applications to transfected HEK-293 cells, we investigated pregnenolone sulfate (PS) effects on deactivation and desensitization of recombinant NMDA receptors subtypes. PS prolonged the deactivation of responses produced by brief applications of L-glutamate with all subunit combinations tested. The action of PS was larger on NR1a/NR2A than on NR1a/NR2B channels. PS slowed the rate of macroscopic desensitization of the responses with all subunit combinations tested. In contrast, PS had little effect on current rise time and had much reduced action on responses with L-cysteate, a low affinity agonist. Our results suggest that PS decreases agonist unbinding. However, this action is counteracted by decreased desensitization. Since desensitization produces slow deactivating components, particularly with NR1a/NR2B receptors, this underlies the decreased PS effect with these subtypes. Indeed PS action was mainly observed on the fast component of deactivation. Furthermore, prolongation of NR1a/NR2A responses was similar to that of responses from NR1b/NR2B receptor, a subtype characterized by reduced desensitization. PS prolongation of evoked NMDA receptor mediated synaptic currents from cortical neuronal primary culture(s) was not significantly different from that of responses with NR1a/NR2B receptors indicating that native receptors in these neurons comprised at least some heteromeric combinations of these two subunits.

Increased exon 5 expression alters extrasynaptic NMDA receptors in cerebellar neurons.

We investigated the ontogenic changes in expression of alternatively spliced forms of NR1 protein that contain the N1 cassette (exon 5) in comparison with the total population of NR1 within the rat cerebellum. The N1 cassette is strongly developmentally regulated in the cerebellum, with >80% of total NR1 protein in the adult rat containing the N1 cassette. In contrast, early in development, <20% of NR1 protein contained this cassette. Rat cortices from the same ages did not show an increase in the percent of NR1 protein expressing the N1 cassette, indicating that the developmental changes in the cerebellum are tissue-specific. As the N1 cassette is known to determine NMDA receptor properties, including spermine sensitivity and decay kinetics of glutamate-induced currents, changes in the characteristics of glutamate-activated currents in granule cells from cerebellar slices were compared at postnatal day 7 versus 14. Glutamate responses exhibited fast deactivation kinetics and reduced potentiation by spermine at day 14 while maintaining sensitivity to an NR2B-selective antagonist. These data are consistent with the possibility that N1 cassette expression may be a factor in the developmental changes in properties of extrasynaptic NMDA receptors in the cerebellum.

A slow NMDA channel: in search of a role.

Recombinant NMDA receptors comprising NR1/NR2D subunits have unique properties when compared with other subtypes. These include low channel conductance with a characteristic pattern of transition between subconductance states and reduced sensitivity to Mg2+ blockade. Native NMDA receptors matching the low conductance and Mg2+ sensitivity of NR1/NR2D recombinant channels have been found in a few neuronal populations, in particular in developing Purkinje neurons (Momiyama et al. 1996). Ultra-rapid agonist application by means of a piezoelectric translator to excised outside-out membrane patches allows concentration jumps in the milliseconds range similar to those occurring during vesicular synaptic release. The high affinity of glutamate for NR1/NR2D receptors produces an extremely slow channel deactivation in response to brief glutamate applications (Monyer et al. 1994). This property has now been identified for the first time by Misra et al. (this issue of The Journal of Physiology, Misra et al. 2000b) for native NMDA channels in mammalian neurons.

Exon 5 and spermine regulate deactivation of NMDA receptor subtypes.

Deactivation of N-methyl-D-aspartate (NMDA) channels after brief agonist exposure determines the duration of their synaptic activation during excitatory neurotransmission. We performed patch-clamp recordings of L-glutamate responses from human embryonic kidney tumoral cells (HEK293) expressing NR1 subunit variants lacking exon 5 together with the NR2B subunit. These responses had deactivation components that lasted several seconds. The presence of exon 5 or spermine greatly accelerated deactivation of L-glutamate responses through alterations in desensitization. These effects were also observed at positive holding potentials and in the presence of physiological Mg(2+). Thus NR1 splicing and polyamines may have profound effects on the kinetics of NMDA receptor-mediated synaptic transmission.

Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons.

In rat cerebellar granule neurons, mRNA and protein levels of the NR2A and NR2C subunits of the NMDA receptor increase during the second postnatal week. At this time, mRNA and protein levels of the NR2B subunit begin to fall. To investigate targeting of NMDA receptor subunits, we performed whole-cell recordings from rat cerebellar granule neurons at different times during development and investigated the pharmacological and biophysical properties of mossy fiber-evoked NMDA EPSCs. Isolated NMDA EPSCs from newly formed synapses in the first postnatal week exhibited partial block by the NR2B subunit-specific antagonist (1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP 101,606). By the end of the second postnatal week, NMDA EPSCs were virtually unaffected by the NR2B antagonist. In parallel, NMDA EPSC decay times decreased over a similar developmental time course. We compared properties of synaptic NMDA receptors with extrasynaptic receptors that are present on the cell body with rapid application of glutamate to excised nucleated patches. Deactivation of patch responses accelerated with development and closely resembled evoked NMDA EPSCs in rats of the same age. However, patch responses were highly sensitive to CP 101,606 through the second postnatal week, and sensitivity was seen in some neurons up to the fourth postnatal week. Spermine potentiated peak NMDA patch responses from postnatal days 10-14 rats but had little effect on evoked NMDA EPSCs. Our data suggest selective targeting of a distinct NMDA receptor subtype to synaptic receptor populations in cerebellar granule neurons. Later in development, similar changes occur in the extrasynaptic receptor population.

Automated serum chemical analysis in the foal.

Blood was collected from healthy Quarter Horse and Appaloosa foals at birth and at intervals until 18 weeks of age and then was processed in an automated system for serum chemical analysis, grouping the results by age. The test values were markedly different from those normally expected in adult horses. The greatest differences were in alkaline phosphatase, lactate dehydrogenase, glucose, and total bilirubin measurements. It was concluded that serum biochemical test results be compared with age-specific normal values before diagnoses are made in cases of illness.

Urinary production in the healthy horse and in horses deprived of feed and water.

Total daily 24-hour urinary output was obtained from 11 healthy horses fed alfalfa hay with free access to salt during periods of high environmental temperatures. Daily urinary volume averaged 15.6 L, with mean specific gravity of 1.028, osmolality of 1,040 mOsm/kg, and urinary flow rate of 1.24 ml/kg/hr. Total 24-hour sample collections of urine were also obtained from horses held without access to feed or water for periods of 24, 48, and 72 hours during high environmental temperatures. Average urine production under these conditions was 6.3 L during the 1st day; 3.2 L, the 2nd day; and 3.0 L, the 3rd day. Although total daily urinary electrolyte excretion decreased during these periods, urinary electrolyte concentrations of the major ions often remained unchanged, or in the case of sodium, actually increased. There was a highly significant correlation between the urinary excretion of the principal cations, sodium and potassium, and the principal anion, chloride. The urinary excretion of water and electrolytes appeared to remain stable after 48 hours of feed and water deprivation.

Clinicopathologic effects of rapid infusion of 5% sodium bicarbonate in 5% dextrose in the horse.

Clinicopathologic effects of rapid intravenous infusion of 3 L of 5% dextrose in water containing 150 g of sodium bicarbonate were evaluated in 8 clinically normal horses. A highly significant metabolic alkalosis was produced in all the horses. This response was maximal at the end of the 20-minute infusion but persisted for as long as 8 hours. Packed cell volume, total plasma proteins, plasma potassium, and plasma chloride concentration decreased significantly after infusion, while plasma sodium concentration increased significantly. The clinical and clinicopathological responses of the horses were comparable to those in man and other animal species.

Physiologic alterations in the horse produced by food and water deprivation during periods of high environmental temperatures.

Eight normal horses were held without access to food or water for 72 hours during a period of high environmental temperatures. During this period, the horses had an average weight loss of 51.6 kg (10.7% of body weight). Highly significant (P less than 0.001) decreases in extracellular fluid volume (18.6 L) and plasma volume (5 L) were observed during this period as compared with base-line values. Plasma protein, sodium, chloride, and osmolality progressively increased in response to the dehydration, whereas packed cell volume, plasma potassium, calcium, magnesium, and phosphate were not significantly altered. The dehydration and clinicopathologic alterations produced were similar to those observed in other species in which the principal problem was a water deficit. After the end of the experimental dehydration period, the horses replaced 62% of the weight loss during a 1-hour period with access to water only.

Volume dilution of sodium thiocyanate as a measure of extracellular fluid volume in the horse.

Volume dilution of sodium thiocyanate as a measure of extracellular fluid volume was determined in 24 normal adult horses and 4 ponies. The method employed yielded highly reproducible results on multiple determinations in individual horses. A highly significant (P less than .001) correlation between thiocyanate space and body weight was demonstrated.

Internal abdominal abscesses in the horse: a study of 25 cases.

internal abdominal abscesses in 25 horses, including 8 horses that died or were euthanatized, were found to be caused by Streptococcus equi, Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis. Breed or sex predilection was not found. Although horses of all ages were affected, those under 5 years of age were more commonly affected. Nearly all of the horses had a history of respiratory catarrh or lymphadenitis. Horses with internal abdominal abscesses also had intermittent, prolonged colic or chronic weight loss. Most horses had increased rectal temperature, increased heart and respiratory rates, and varying degrees of anorexia. Chronic depression anemia, increased plasma fibrinogen concentration, increased total plasma protein with hypergammaglobulinemia, and hypoalbuminemia were prominent clinicopathologic findings. Results of rectal examination or surgical exploration often revealed an abdominal mass. Peritoneal fluid from 14 horses was examined, and the protein content was greater than 2.5 g/dl, with increased numbers of neutrophils. Culture results were negative in all 14 cases, although bacteria were seen intracellularly in 5 cases. Long-term (2- to 6-month) antimicrobial therapy with procaine penicillin G resulted in clinical resolution in most of these animals.

Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with the zinc sulfate turbidity test, serum electrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test.

Four procedures for assessment of adequacy of colostral immunoglobulin (Ig) transfer in foals were evaluated. Results of zinc sulfate turbidity test, serum electrophoresis, total serum protein refractometry, and sodium sulfite precipitation test were compared with immunoglobulin G content determined by single radial immunodiffusion. The zinc sulfate turbidity test gave acceptable results for IgG, except that hemolyzed serum samples gave higher than expected values. A correction factor for hemolyzed serum was found to be useful. Serum electrophoresis was a satisfactory method of estimating IgG content. Total serum protein values may not be a valid basis for estimating IgG content, inasmuch as postsuckling total protein values were found to decrease in some foals in which passive transfer of IgG had been adequate. Sodium sulfite precipitation reactions were too unpredictable to be of value for determination of neonatal IgG concentration.

Disseminated septic meningitis in a mare.

Physical, neurologic, and clinicopathologic findings in a 3-year-old female Morgan horse with anorexia and nasal discharge suggested suppurative meningitis. Necropsy findings substantiated the tentative diagnosis. Actinomyces sp was cultured from a meningeal abscess surrounding the pituitary gland and from resolving lung abscesses.