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Learning and memory are thought to require the induction and maintenance of long-term potentiation (LTP) of synaptic strength. LTP induction requires the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) but for structural rather than enzymatic functions. We show that the relevant structural function is regulated by CaMKII binding to the NMDA-type glutamate receptor subunit GluN2B. This binding directly generates Ca 2+ -independent autonomous CaMKII activity, and we show that this enzymatic activity is dispensable for LTP induction (within 5 min) but required for a subsequent LTP phase (within 15 min). This requirement for CaMKII activity provides an objective temporal definition for the intermediary phase of LTP expression. Later LTP maintenance may still require structural functions of GluN2B-bound CaMKII but not the resulting enzymatic CaMKII activity or their co-condensation. Thus, autonomous CaMKII activity mediates post-induction LTP but (1) via GluN2B binding, not T286 autophosphorylation, and (2) during the intermediary expression phase rather than for long-term maintenance., Competing Interests: Declaration of interests The Regents of the University of Colorado have filed a patent application, with N.L.R., M.E.L., J.E.T., S.J.C., and K.U.B. in the list of inventors, that includes findings reported in this manuscript. K.U.B. is co-founder and board member of Neurexis Therapeutics, a company that seeks to develop a CaMKII inhibitor into a therapeutic drug for cerebral ischemia., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuronal excitatory synapses are primarily located on small dendritic protrusions called spines. During synaptic plasticity underlying learning and memory, Ca 2+ influx through postsynaptic NMDA-type glutamate receptors (NMDARs) initiates signaling pathways that coordinate changes in dendritic spine structure and synaptic function. During long-term potentiation (LTP), high levels of NMDAR Ca 2+ influx promote increases in both synaptic strength and dendritic spine size through activation of Ca 2+ -dependent protein kinases. In contrast, during long-term depression (LTD), low levels of NMDAR Ca 2+ influx promote decreased synaptic strength and spine shrinkage and elimination through activation of the Ca 2+ -dependent protein phosphatase calcineurin (CaN), which is anchored at synapses via the scaffold protein A-kinase anchoring protein (AKAP)150. In Alzheimer's disease (AD), the pathological agent amyloid-β (Aβ) may impair learning and memory through biasing NMDAR Ca 2+ signaling pathways toward LTD and spine elimination. By employing AKAP150 knock-in mice of both sexes with a mutation that disrupts CaN anchoring to AKAP150, we revealed that local, postsynaptic AKAP-CaN-LTD signaling was required for Aβ-mediated impairment of NMDAR synaptic Ca 2+ influx, inhibition of LTP, and dendritic spine loss. Additionally, we found that Aβ acutely engages AKAP-CaN signaling through activation of G-protein-coupled metabotropic glutamate receptor 1 (mGluR1) leading to dephosphorylation of NMDAR GluN2B subunits, which decreases Ca 2+ influx to favor LTD over LTP, and cofilin, which promotes F-actin severing to destabilize dendritic spines. These findings reveal a novel interplay between NMDAR and mGluR1 signaling that converges on AKAP-anchored CaN to coordinate dephosphorylation of postsynaptic substrates linked to multiple aspects of Aβ-mediated synaptic dysfunction., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) 1-3 . However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP 4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B 9-14 . Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked., (© 2023. The Author(s).)

CaMKII has molecular memory functions because transient calcium ion stimuli can induce long-lasting increases in its synaptic localization and calcium ion-independent (autonomous) activity, thereby leaving memory traces of calcium ion stimuli beyond their duration. The synaptic effects of two mechanisms that induce CaMKII autonomy are well studied: autophosphorylation at threonine-286 and binding to GluN2B. Here, we examined the neuronal functions of additional autonomy mechanisms: nitrosylation and oxidation of the CaMKII regulatory domain. We generated a knock-in mouse line with mutations that render the CaMKII regulatory domain nitrosylation/oxidation-incompetent, CaMKII ΔSNO , and found that it had deficits in memory and synaptic plasticity that were similar to those in aged wild-type mice. In addition, similar to aged wild-type mice, in which CaMKII was hyponitrosylated, but unlike mice with impairments of other CaMKII autonomy mechanisms, CaMKII ΔSNO mice showed reduced long-term potentiation (LTP) when induced by theta-burst stimulation but not high-frequency stimulation (HFS). As in aged wild-type mice, the HFS-LTP in the young adult CaMKII ΔSNO mice required L-type voltage-gated calcium ion channels. The effects in aged mice were likely caused by the loss of nitrosylation because no decline in CaMKII oxidation was detected. In hippocampal neurons, nitrosylation of CaMKII induced its accumulation at synapses under basal conditions in a manner mediated by GluN2B binding, like after LTP stimuli. However, LTP-induced synaptic CaMKII accumulation did not require nitrosylation. Thus, an aging-associated decrease in CaMKII nitrosylation may cause impairments by chronic synaptic effects, such as the decrease in basal synaptic CaMKII.

Genetic alterations in autism spectrum disorders (ASD) frequently disrupt balance between synaptic excitation and inhibition and alter plasticity in the hippocampal CA1 region. Individuals with Timothy Syndrome (TS), a genetic disorder caused by Ca V 1.2 L-type Ca 2+ channel (LTCC) gain-of function mutations, such as G406R, exhibit social deficits, repetitive behaviors, and cognitive impairments characteristic of ASD that are phenocopied in TS2-neo mice expressing G406R. Here, we characterized hippocampal CA1 synaptic function in male TS2-neo mice and found basal excitatory transmission was slightly increased and inhibitory transmission strongly decreased. We also found distinct impacts on two LTCC-dependent forms of long-term potentiation (LTP) synaptic plasticity that were not readily consistent with LTCC gain-of-function. LTP induced by high-frequency stimulation (HFS) was strongly impaired in TS2-neo mice, suggesting decreased LTCC function. Yet, Ca V 1.2 expression, basal phosphorylation, and current density were similar for WT and TS2-neo. However, this HFS-LTP also required GABA A receptor activity, and thus may be impaired in TS2-neo due to decreased inhibitory transmission. In contrast, LTP induced in WT mice by prolonged theta-train (PTT) stimulation in the presence of a β-adrenergic receptor agonist to increase Ca V 1.2 phosphorylation was partially induced in TS2-neo mice by PTT stimulation alone, consistent with increased LTCC function. Overall, our findings provide insights regarding how altered Ca V 1.2 channel function disrupts basal transmission and plasticity that could be relevant for neurobehavioral alterations in ASD., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Previously, we found that amyloid-beta (Aβ) competitively inhibits the kinesin motor protein KIF11 (Kinesin-5/Eg5), leading to defects in the microtubule network and in neurotransmitter and neurotrophin receptor localization and function. These biochemical and cell biological mechanisms for Aβ-induced neuronal dysfunction may underlie learning and memory defects in Alzheimer's disease (AD). Here, we show that KIF11 overexpression rescues Aβ-mediated decreases in dendritic spine density in cultured neurons and in long-term potentiation in hippocampal slices. Furthermore, Kif11 overexpression from a transgene prevented spatial learning deficits in the 5xFAD mouse model of AD. Finally, increased KIF11 expression in neuritic plaque-positive AD patients' brains was associated with better cognitive performance and higher expression of synaptic protein mRNAs. Taken together, these mechanistic biochemical, cell biological, electrophysiological, animal model, and human data identify KIF11 as a key target of Aβ-mediated toxicity in AD, which damages synaptic structures and functions critical for learning and memory in AD., Competing Interests: The authors have no financial conflicts of interest., (© 2022 The Authors.)

Regulated insertion and removal of postsynaptic AMPA glutamate receptors (AMPARs) mediates hippocampal long-term potentiation (LTP) and long-term depression (LTD) synaptic plasticity underlying learning and memory. In Alzheimer's disease β-amyloid (Aβ) oligomers may impair learning and memory by altering AMPAR trafficking and LTP/LTD balance. Importantly, Ca 2+ -permeable AMPARs (CP-AMPARs) assembled from GluA1 subunits are excluded from hippocampal synapses basally but can be recruited rapidly during LTP and LTD to modify synaptic strength and signaling. By employing mouse knockin mutations that disrupt anchoring of the kinase PKA or phosphatase Calcineurin (CaN) to the postsynaptic scaffold protein AKAP150, we find that local AKAP-PKA signaling is required for CP-AMPAR recruitment, which can facilitate LTP but also, paradoxically, prime synapses for Aβ impairment of LTP mediated by local AKAP-CaN LTD signaling that promotes subsequent CP-AMPAR removal. These findings highlight the importance of PKA/CaN signaling balance and CP-AMPARs in normal plasticity and aberrant plasticity linked to disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reduced α7 nicotinic acetylcholine receptor (nAChR) function is linked to impaired hippocampal-dependent sensory processing and learning and memory in schizophrenia. While knockout of the Chrna7 gene encoding the α7nAChR on a C57/Bl6 background results in changes in cognitive measures, prior studies found little impact on hippocampal synaptic plasticity in these mice. However, schizophrenia is a multi-genic disorder where complex interactions between specific genetic mutations and overall genetic background may play a prominent role in determining phenotypic penetrance. Thus, we compared the consequences of knocking out the α7nAChR on synaptic plasticity in C57/Bl6 and C3H mice, which differ in their basal α7nAChR expression levels. Homozygous α7 deletion in C3H mice, which normally express higher α7nAChR levels, resulted in impaired long-term potentiation (LTP) at hippocampal CA1 synapses, while C3H α7 heterozygous mice maintained robust LTP. In contrast, homozygous α7 deletion in C57 mice, which normally express lower α7nAChR levels, did not alter LTP, as had been previously reported for this strain. Thus, the threshold of Chrna7 expression required for LTP may be different in the two strains. Measurements of auditory gating, a hippocampal-dependent behavioral paradigm used to identify schizophrenia-associated sensory processing deficits, was abnormal in C3H α7 knockout mice confirming that auditory gating also requires α7nAChR expression. Our studies highlight the importance of genetic background on the regulation of synaptic plasticity and could be relevant for understanding genetic and cognitive heterogeneity in human studies of α7nAChR dysfunction in mental disorders., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aβ) 1-42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, includingN-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aβ(1-42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aβ-induced synaptic dysfunction, but we recently found that Aβ(1-42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aβ(1-42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aβ, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aβ(1-42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aβ(1-42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5-1.0μM Aβ(1-42) or 1-5μM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aβon LTP and synapse loss, suggesting that Aβinduces acute and chronic synaptic dysfunction in part through inhibiting Eg5., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Neuroinflammation is a component of secondary injury following traumatic brain injury (TBI) that can persist beyond the acute phase. Leukotrienes are potent, pro-inflammatory lipid mediators generated from membrane phospholipids. In the absence of injury, leukotrienes are undetectable in the brain, but after trauma they are rapidly synthesized by a transcellular event involving infiltrating neutrophils and endogenous brain cells. Here, we investigate the efficacy of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP), in blocking leukotriene synthesis, secondary brain damage, synaptic dysfunction, and cognitive impairments after TBI. Male Sprague Dawley rats (9-11weeks) received either MK-886 or vehicle after they were subjected to unilateral moderate fluid percussion injury (FPI) to assess the potential clinical use of FLAP inhibitors for TBI. MK-886 was also administered before FPI to determine the preventative potential of FLAP inhibitors. MK-886 given before or after injury significantly blocked the production of leukotrienes, measured by reverse-phase liquid chromatography coupled to tandem mass spectrometry (RP LC-MS/MS), and brain edema, measured by T2-weighted magnetic resonance imaging (MRI). MK-886 significantly attenuated blood-brain barrier disruption in the CA1 hippocampal region and deficits in long-term potentiation (LTP) at CA1 hippocampal synapses. The prevention of FPI-induced synaptic dysfunction by MK-886 was accompanied by fewer deficits in post-injury spatial learning and memory performance in the radial arm water maze (RAWM). These results indicate that leukotrienes contribute significantly to secondary brain injury and subsequent cognitive deficits. FLAP inhibitors represent a novel anti-inflammatory approach for treating human TBI that is feasible for both intervention and prevention of brain injury and neurologic deficits., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and other kinases, whereas long-term depression (LTD) requires phosphatases. Here, we found that LTD also requires CaMKII and its phospho-T286-induced "autonomous" (Ca(2+)-independent) activity. However, whereas LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by "autonomous" CaMKII was further stimulated by Ca(2+)/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), whereas GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

This commentary discusses the important contributions of the article published in this journal by Huang and colleagues, titled, "Acute ethanol exposure increases firing and induces oscillations in cerebellar Golgi cells of freely moving rats." In this manuscript, Huang and colleagues present a number of interesting and important findings. While it has been shown previously that ethanol (EtOH) causes an increase in the firing of cerebellar Golgi cells in brain slice preparations and anesthetized animals, here the authors provide the first evidence that this action of EtOH occurs in vivo in freely moving, unanesthetized animals. These results also enhance our understanding of cerebellar functioning by describing the mechanism by which EtOH essentially de-afferentates (blocks specific inputs to) the cerebellum from the normal processing of sensory signals due to EtOH-induced Golgi neuron excitation, resulting in inhibition of granule cells. Furthermore, the authors characterize the novel observation of EtOH-induced neuronal oscillations, which was not previously observed in other preparations., (Copyright © 2012 by the Research Society on Alcoholism.)

AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca(2+) influx. However, a small number of Ca(2+)-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca(2+) signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca(2+)-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca(2+)-permeable AMPARs both basally and during LTP and LTD.

Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) "autonomy" (T286-autophosphorylation-induced Ca(2+)-independent activity) is required for long-term potentiation (LTP) and for learning and memory, as demonstrated by CaMKII T286A mutant mice. The >20-year-old hypothesis that CaMKII stimulation is required for LTP induction, while CaMKII autonomy is required for LTP maintenance was recently supported using the cell-penetrating fusion-peptide inhibitor antCN27. However, we demonstrate here that ant/penetratin fusion to CN27 compromised CaMKII-selectivity, by enhancing a previously unnoticed direct binding of CaM to ant/penetratin. In contrast to antCN27, the improved cell-penetrating inhibitor tatCN21 (5 mum) showed neither CaM binding nor inhibition of basal synaptic transmission. In vitro, tatCN21 inhibited stimulated and autonomous CaMKII activity with equal potency. In rat hippocampal slices, tatCN21 inhibited LTP induction, but not LTP maintenance. Correspondingly, tatCN21 also inhibited learning, but not memory storage or retrieval in a mouse in vivo model. Thus, CaMKII autonomy provides a short-term molecular memory that is important in the signal computation leading to memory formation, but is not required as long-term memory store.

Recent evidence demonstrates that N-methyl-d-aspartate receptor (NMDAR) trafficking contributes to synaptic plasticity in the hippocampus. Phosphorylation of tyrosine residues, especially NR2B tyrosine 1472, appears to be a mechanism by which NMDAR endocytosis is prevented, suggesting that the tyrosine phosphorylation and surface expression of NMDARs are positively correlated. Previous work from our laboratory and others has confirmed that modulation of tyrosine phosphatase and kinase activity alters the surface expression of NMDARs. However, the changes in NMDAR surface expression described in those studies were in terms of total surface membrane versus intracellular receptors. Within the plasma membrane of glutamatergic synapses, distinct populations of NMDARs exist. Namely, receptors at the surface can be differentiated into synaptic and extrasynaptic pools based on their association with the post-synaptic density (PSD) and availability to glutamate. In the present study, we utilized a subcellular fractionation approach coupled with detergent extraction to prepare synaptic and extrasynaptic NMDARs from adult rat hippocampal slices. Using this method, we examined how tyrosine phosphatase and Src-family tyrosine kinase (SFK) inhibitors modulate the phosphorylation and localization of these different pools of NMDARs. We found that both synaptic and extrasynaptic NMDARs were modulated by tyrosine phosphatase and SFK inhibitors; however subunit- and residue-specific effects were observed. Specifically, phosphorylation of NR2B tyrosine 1472 was associated with enrichment of synaptic NMDARs, whereas phosphorylation of NR2B tyrosine 1336 was associated with enrichment of extrasynaptic NMDARs. Using electrophysiological methods, we also reveal that the biochemical modifications produced by these inhibitors were associated with corresponding changes in NMDAR function.

This study was designed to examine the neuronal mechanisms of ethanol sensitivity by utilizing inbred short sleep (ISS) and inbred long sleep (ILS) mouse strains that display large differences in sensitivity to the behavioural effects of ethanol. Comparisons of whole-cell electrophysiological recordings from CA1 pyramidal neurons in hippocampal slices of ISS and ILS mice indicate that ethanol enhances GABAA receptor-mediated inhibitory postsynaptic currents (GABAA IPSCs) and reduces NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) in a concentration- and strain-dependent manner. In ILS neurons, these receptor systems are significantly more sensitive to ethanol than those in ISS neurons. To further examine the underlying mechanisms of differential ethanol sensitivities in these mice, GABAB activity and presynaptic and postsynaptic actions of ethanol were investigated. Inhibition of GABAB receptor function enhances ethanol-mediated potentiation of distal GABAA IPSCs in ILS but not ISS mice, and this blockade of GABAB receptor function has no effect on the action of ethanol on NMDA EPSCs in either mouse strain. Thus, subregional differences in GABAB activity may contribute to the differential ethanol sensitivity of ISS and ILS mice. Moreover, analysis of the effects of ethanol on paired-pulse stimulation, spontaneous IPSC events, and brief local GABA or glutamate application suggest that postsynaptic rather than presynaptic mechanisms underlie the differential ethanol sensitivity of these mice. Furthermore, these results provide essential information to focus better on appropriate target sites for more effective drug development for the treatment of alcohol abuse.

High-alcohol-sensitive (HAS) and low-alcohol-sensitive (LAS) rats were bred for sensitivity and insensitivity, respectively, to the sedative/hypnotic effects of ethanol. These rats also display differential sensitivity to the depressant effects of locally applied ethanol on cerebellar Purkinje neurons in vivo. We have found that LAS animals exhibit a greater influence of endogenous beta-adrenergic activity on neuronal responses to gamma-aminobutyric acid (GABA) and ethanol than do HAS animals. In the current study, we investigated the possibility that the regulation of synaptic norepinephrine levels by norepinephrine transporters could contribute to a differential beta-adrenergic influence on GABA and ethanol sensitivity between HAS and LAS rats. We locally applied norepinephrine from a glass micropipette into the various layers of cerebellar brain slices prepared from LAS and HAS rats, and recorded the levels of norepinephrine clearance by using Nafion-coated carbon-fiber microelectrodes. Norepinephrine clearance was significantly faster by approximately 64% in the Purkinje cell layer of HAS rats. No differences in norepinephrine clearance were found in the molecular or the granule layer between LAS and HAS rats. The catecholamine uptake inhibitor nomifensine reduced norepinephrine clearance in both rat lines. These findings support the hypothesis that regulation of synaptic norepinephrine levels by norepinephrine transporter activity in the Purkinje cell layer may contribute to the differential sensitivity of Purkinje neurons to ethanol and GABA in LAS and HAS rats.

Parkinson's disease (PD) is characterized by a degeneration of the dopamine (DA) pathway from the substantia nigra (SN) to the basal forebrain. Prior studies in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats have primarily concentrated on the implantation of fetal ventral mesencephalon (VM) into the striatum in attempts to restore DA function in the target. We implanted solid blocks of fetal VM or fetal striatal tissue into the SN to investigate whether intra-nigral grafts would restore motor function in unilaterally 6-OHDA-lesioned rats. Intra-nigral fetal striatal and VM grafts elicited a significant and long-lasting reduction in apomorphine-induced rotational behavior. Lesioned animals with ectopic grafts or sham surgery as well as animals that received intra-nigral grafts of fetal cerebellar cortex showed no recovery of motor symmetry. Subsequent immunohistochemical studies demonstrated that VM grafts, but not cerebellar grafted tissue expressed tyrosine hydroxylase (TH)-positive cell bodies and were associated with the innervation by TH-positive fibers into the lesioned SN as well as adjacent brain areas. Striatal grafts were also associated with the expression of TH-positive cell bodies and fibers extending into the lesioned SN and an induction of TH-immunolabeling in endogenous SN cell bodies. This finding suggests that trophic influences of transplanted fetal striatal tissue can stimulate the re-expression of dopaminergic phenotype in SN neurons following a 6-OHDA lesion. Our data support the hypothesis that a dopaminergic re-innervation of the SN and surrounding tissue by a single solid tissue graft is sufficient to improve motor asymmetry in unilateral 6-OHDA-lesioned rats.

We previously found that beta adrenergic agonists such as norepinephrine and isoproterenol potentiate the depressant actions of ethanol (EtOH) on cerebellar Purkinje neurons. Furthermore, antagonism of the beta adrenergic effects of endogenously released catecholamines with timolol reduced EtOH-induced depressions of neuronal activity in that brain area. In the present study, we further investigated the hypothesis that activity of the endogenous noradrenergic innervation to the cerebellar cortex can potentiate this EtOH action. We investigated the interaction of synaptically released catecholamines on EtOH-induced depressions of cerebellar Purkinje neurons in three different experiments: (1) endogenous catecholamine release was facilitated by applying the catecholamine uptake inhibitor desmethylimipramine, (2) activity of the noradrenergic innervation of the cerebellar cortex from locus ceruleus was increased by causing acute withdrawal from 7 days of chronic morphine treatment with the opiate antagonist naloxone, and (3) the noradrenergic innervation of the cerebellum was activated directly by electrical stimulation of the locus ceruleus. We found that all three conditions potentiated EtOH-induced depressions in the cerebellum and that this potentiation of ethanol effects could be antagonized by the systemic administration of the beta adrenergic antagonist propranolol. Furthermore, morphine withdrawal also caused potentiation of the depressant effects of phencyclidine, which are known to be regulated by the endogenous catecholamine innervation in this brain area. Taken together with our previous data demonstrating a beta adrenergic facilitation of EtOH actions in this brain area, the present results suggest that the activity of endogenous noradrenergic synapses can regulate the depressant effects of EtOH on cerebellar Purkinje neurons.

Fetal spinal cord tissue grafts have been advocated as a possible repair strategy for spinal cord injury. In the present study, we used intraocular spinal cord grafts to model the interactions which may occur between fetal and adult spinal cord after making such a graft and to study to which extent functional connections can be expected to occur between the host and graft tissue. We first grafted fetal spinal cord to the anterior chamber of the eye where it was allowed to mature. A second piece of fetal spinal cord was then sequentially grafted in contact with the first graft. Electrophysiological recordings made from the older graft while electrically stimulating the younger graft provided evidence for an excitatory innervation from the younger spinal cord graft to the mature spinal cord which appeared to be glutamatergic. However, we only rarely found excitatory inputs from the first, mature spinal cord graft to the younger graft. Fiber connections between the two spinal cord grafts were verified by retrograde tracing and neurofilament immunohistochemistry. In no case was a trophic influence on graft volume observed between spinal cord grafts regardless of whether the transplantations were performed sequentially or at the same time. Even the introduction of a second graft to immature spinal cord tissue was ineffective. In contrast, we found a marked trophic, neuron-rescuing effect of spinal cord grafts upon cografts of fetal dorsal root ganglia. This latter observation is consistent with the hypothesis that spinal cord tissue can exert a trophic effect on developing sensory ganglia and demonstrates that many sensory neurons can survive in the presence of a central target and in the absence of the appropriate peripheral target. These intraocular experiments predict that fetal spinal cord grafted to the injured adult spinal cord may develop effective excitatory inputs with the host, while host-to-graft inputs may develop to a considerably smaller extent. Our results also suggest that the adult spinal cord does not exert marked trophic effects on growth of fetal spinal cord, while it does exert a trophic influence on central projections of dorsal root ganglia.

Previous studies have found that ethanol (EtOH) will consistently potentiate gamma-aminobutyric acid (GABA) receptor function in the cerebellum during beta adrenergic receptor activation. One consequence of beta adrenergic receptor stimulation is to increase cAMP levels, which, in turn, activate protein kinase A (PKA)-mediated phosphorylation of intracellular protein sites. In the present study, we investigated three cAMP analogues, two activators and one inhibitor of PKA to determine whether this cAMP-mediated second-messenger system may be one mechanism involved in the previously observed beta adrenergic interaction of EtOH with the GABA(A) receptor. Furthermore, because the phosphorylation state of the GABA(A) receptor may be an important determinant of function, we investigated the effect of the block of phosphatase activity on EtOH/GABA receptor interactions. We found that similar to the beta adrenergic agonist isoproterenol, local applications of the membrane-permeable cAMP analogues 8-bromo-cAMP and Sp-cAMP could modulate responses to iontophoretically applied GABA and that these modulated GABA responses were sensitized to the potentiative effects of EtOH. EtOH did not facilitate unmodulated GABA effects or GABA responses that were maximally modulated by 8-bromo-cAMP, suggesting that the cAMP mechanism mediates the observed EtOH interaction with GABA mechanisms. Furthermore, the PKA inhibitor Rp-cAMP reversed the EtOH-induced potentiation of the isoproterenol-modulated GABA responses. Finally, microcystin-LR and okadaic acid, which are type I and IIa phosphatase inhibitors, could also modulate and sensitize GABA responses to EtOH. These data suggest that beta adrenergic sensitization of GABA(A) receptors to EtOH involves the intracellular cAMP/PKA second-messenger cascade.

Rapid acute neuronal tolerance (RANT) to the depressant effects of ethanol (EtOH) is a desensitization of EtOH-induced depression of neuronal firing that develops over the first 5 to 7 min of EtOH exposure. This phenomenon has been hypothesized to play a role in acute behavioral insensitivity to EtOH and is expressed by cerebellar Purkinje neurons in animals selectively bred for insensitivity to EtOH-induced ataxia, such as low-alcohol-sensitive (LAS) rats and short-sleep mice. Purkinje neurons of animals bred for high sensitivity to EtOH-induced behavioral ataxia, such as high-alcohol-sensitive (HAS) rats and long-sleep mice, only infrequently express such acute tolerance to EtOH-induced depression of neuronal activity. However, because higher EtOH doses are required to depress Purkinje neuron activity in LAS rats than in HAS rats, it was not known whether the higher EtOH doses that depress LAS neurons would also induce RANT to EtOH in HAS rats, which were generally not exposed to such high EtOH doses in previous studies. Furthermore, the conditions for development and maintenance of RANT to EtOH had not been characterized. We found that RANT to EtOH-induced depression of cerebellar neurons principally developed within 5 min of EtOH application and recovered within 20 min of the last EtOH exposure and that neurons in HAS rats did not develop acute tolerance to the higher EtOH doses that were effective in LAS rats. We conclude that this rapid tolerance contributes to the acute EtOH sensitivity difference between LAS and HAS rats.

Local application of ethanol (EtOH) has been reported to inhibit Purkinje neuron firing. EtOH-induced depressions can be antagonized by bicuculline, suggesting involvement of GABAA receptors. Since there is evidence from other studies indicating that nicotine may interact with EtOH responses, in this study we investigated whether nicotinic acetylcholine receptors (nAChR's) might be also involved in EtOH-induced depressions of these neurons in urethane-anesthetized Sprague-Dawley rats. Using local application (micropressure ejection) of drugs onto cerebellar Purkinje neurons while recording extracellular firing rates, we found that depressant responses to EtOH could be potentiated by subdepressant doses of nicotine. Furthermore, EtOH-induced depressions of firing could be antagonized by mecamylamine, a nicotinic acetylcholine receptor (nAChR) antagonist. Results from the present study indicate that EtOH-induced depressions may involve nAChRs in the cerebellum.

Previous studies in our laboratory indicated that electrophysiological responses of cerebellar Purkinje neurons to GABA were not routinely potentiated by ethanol (EtOH), and the potentiation was not large when it occurred. In the presence of beta-adrenergic agonists, such as isoproterenol, however, GABA inhibitions became sensitive to potentiation by EtOH in nearly every Purkinje neuron tested. beta-adrenergic receptor activation alone also modulates (potentiates) GABA responses on Purkinje neurons, and this has been reported to be mediated by a cAMP second messenger system. Herein, we report that the membrane-permeable cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP), but not the membrane-impermeable cAMP, can also modulate GABA responses and that EtOH potentiates this facilitatory action of 8-Br-cAMP. These effects are not likely caused by adenosine receptor mechanisms, because this 8-bromoadenosine mediated modulation and sensitization was observed in the presence of systemic theophylline. These data suggest that the beta-adrenergic modulation and sensitization to EtOH of cerebellar Purkinje neuron GABA responses occur via a cAMP second messenger mechanism.

Electrophysiological studies indicate that EtOH decreases the firing rate of cerebellar Purkinje neurons in vivo and in vitro through a GABAA mechanism. These neurons receive a prominent noradrenergic input from the locus coeruleus. Stimulation of the locus coeruleus or local application of beta-adrenergic agonists potentiates Purkinje neuron responses to GABA and sensitizes GABA responses to the potentiative effects of EtOH. In the present study, we found that the modulatory influences of the beta-adrenergic agonist isoproterenol potentiated EtOH-induced depressions of Purkinje neuron firing. This isoproterenol interaction with EtOH was antagonized by the beta-adrenergic antagonist timolol. We found evidence that endogenous catecholamines can cause this effect as well. Timolol antagonized EtOH-induced depressions on 20% of the neurons studied. This was the same frequency as that previously found for EtOH-induced potentiations of GABA depressions in this brain area. These data suggest that the Purkinje neurons showing this interaction receive spontaneously active catecholamine inputs that sensitize the GABA effects to the potentiative effects of ethanol. Consistent with this hypothesis, we also found that timolol antagonized this GABA/EtOH interaction. Taken together, these results are consistent with the hypothesis that EtOH-induced depressions of Purkinje neurons involved endogenous GABA actions that may be regulated by beta-adrenergic mechanisms.

Skeletal muscle from embryonic day 20 (E20) was combined with E15 rat spinal cord in the anterior chamber of the eye of adult albino rats. The two grafts were either transplanted concomitantly or sequentially, in which case muscle tissue was added 4 months after the spinal cord. Control groups received a single graft of either spinal cord or skeletal muscle. Survival and intraocular growth were observed through the cornea. After maturation in oculo, the double grafts were examined immunohistologically utilizing antisera to neurofilament (NF) and acetylcholinesterase (AChE). The grafts were also evaluated using electrical stimulation to determine functional connectivity. The spinal cord and skeletal muscle grafts were found to exert reciprocal trophic effects on each other, evidenced as a larger muscle mass in skeletal muscle grafts allowed to develop in the presence of spinal cord tissue, and a larger volume of spinal cord grafts allowed to develop together with a skeletal muscle graft, respectively. Immunohistochemistry revealed NF-positive nerve fibers leaving the spinal cord graft and entering the muscle tissue. AChE-positive endplates developed in the muscle grafts. Electrical stimulation of the spinal cord part of double-graft combinations generally elicited contractile responses in specific areas of the muscle cograft. These results demonstrate both structural and functional connections between grafts of spinal cord and skeletal muscle tissue in vivo. The fact that such connections were also established between a mature (adult) spinal cord graft and fetal skeletal muscle tissue suggests that some alpha-motoneurons are able to survive for many months in the intraocular grafts without an appropriate target, and that they are able to subsequently innervate skeletal muscle targets.

Previous studies have demonstrated that ethanol (EtOH) inhibits the firing rate of Purkinje neurons both in vitro and in vivo. However, little is known about the response of cerebellar interneurons to EtOH. In this report, we describe the effects of locally applied EtOH on the firing of one type of cerebellar interneuron, tentatively identified as Golgi neurons, and on Purkinje cells in brain slices in vitro. The Golgi neurons were excited by EtOH, whereas EtOH depressed the firing rate of Purkinje neurons. To the best of our knowledge, this is the first report of responses of cerebellar Golgi neurons to local applications of EtOH.

Biochemical studies indicate that ethanol (EtOH) will facilitate the activation of the GABAA/Cl- channel, and behavioral studies demonstrate that EtOH-induced sedative and incoordinating effects can be potentiated by GABA mimetics and blocked by GABA antagonists. It has been difficult, however, to demonstrate an EtOH-induced potentiation of the depressant electrophysiological effects of locally applied GABA in mammalian brain in vivo. Similarly, in this study, local EtOH applications only infrequently caused potentiations of the depressant effects of microiontophoretically applied GABA on cerebellar Purkinje neurons, and this interaction was modest when present. The predominant interaction of locally applied EtOH was an antagonism of GABA-induced depressions of neuronal activity. However, the GABAA receptor antagonist bicuculline reversibly and apparently competitively blocked the depressant effects of locally applied EtOH on single cerebellar Purkinje neurons. Our data suggest that EtOH potentiation of GABA responses alone is insufficient to account for EtOH-induced depressions of cerebellar Purkinje neurons. However, these data clearly imply that activation of a GABAA receptor is required for the expression of EtOH-induced depressions of neuronal activity in this brain area. It is less clear how lower, nondepressant doses of EtOH interact with GABA mechanisms. We hypothesize that either the GABAA receptor mechanism must be sensitized to the potentiative effects of EtOH through the influences of neuromodulatory and/or hormonal regulation, or that EtOH interacts directly with these regulatory processes.

We previously reported that both systemic administration and brief local application of ethanol potentiated gamma-aminobutyric acid (GABA)-induced depressions of cerebellar Purkinje neurons when the GABA responses were concomitantly facilitated (modulated) by catecholaminergic agonists. In the present study, we further investigated the effects of prolonged local applications of ethanol, which more closely mimic the systemic application of ethanol, and we characterized the pharmacological specificity of the catecholaminergic interaction with these ethanol effects. As has been previously observed, iontophoretic applications of isoproterenol (ISO), a beta adrenergic agonist, facilitated GABA-induced depressions of cerebellar Purkinje neurons. The prolonged local application of ethanol produced a long-lasting potentiation of the ISO-modulated GABA responses that was similar in duration to that caused by systemic ethanol administration. The ethanol-induced augmentation of the ISO-modulated GABA responses was diminished both by terminating the beta adrenergic agonist application as well as by administering the beta adrenergic antagonist timolol. The alpha adrenergic agonist phenylephrine, on the other hand, either attenuated or had no effects on the GABA-induced depressions of cerebellar Purkinje neurons, and a subsequent application of ethanol did not potentiate GABA responses in the presence of phenylephrine. We conclude that prolonged local application of ethanol mimics the interaction of systemic ethanol with GABA-induced depressions of cerebellar Purkinje neurons. Furthermore, the catecholaminergic sensitization of GABA responses to these potentiative effects of ethanol is mediated by a beta adrenergic mechanism.

In this study, we confirmed that microiontophoretically applied norepinephrine (NE) and isoproterenol potentiate the depressant effects of locally-applied gamma-aminobutyric acid (GABA) on cerebellar Purkinje neurons of anesthetized rats. Although ethanol (EtOH) does not reliably or efficaciously potentiate GABA-induced depressions of neuronal activity, we found that systemic or locally-applied EtOH does markedly potentiate GABA-induced inhibitions of Purkinje neuron firing rate if that response is concomitantly modulated by NE or isoproterenol. This study suggests that the EtOH sensitivity of the GABA mechanism of electrophysiological responses in the cerebellar cortex is regulated by the neuromodulatory effect of beta-adrenergic receptor activation.

Previous studies indicated that the excitatory effects of nicotine may be mediated via interference with GABAergic transmission. Here, several variants of the paired-pulse paradigm were employed to ascertain whether nicotine interferes with endogenous inhibitory circuits in the hippocampus. Nicotine attenuated the inhibition evoked by antidromic (alvear) stimulation in the CA1 region in a concentration-dependent manner (EC50 = 60-75 microM). This same phenomenon was also observed for the GABAA receptor antagonist bicuculline (0.1 microM). Orthodromic-orthodromic paired-pulse paradigms were found to be unsuitable for investigating the effects of epileptogenic agents such as nicotine and bicuculline on endogenous inhibition.

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) blocked seizures induced by nicotine (4 mg/kg, i.p.) in C3H male mice with an ID50 of 2.37 +/- 0.66 mg/kg (average +/- 95% confidence limit). This steroid (1 microM) also increased paired-pulse inhibition in the hippocampus approximately 40% after 50 min exposure; nicotine (200 microM) partially reversed this effect. Since nicotine and 3 alpha-OH-DHP may have opposite effects on endogenous inhibitory systems, it is proposed that nicotine-induced seizures may involve a disinhibitory mechanism and that 3 alpha-OH-DHP protects against seizures by preventing disinhibition.

Previous data indicated that bath-application of nicotine to mouse hippocampal slices resulted in a concentration-dependent increase in the amplitude of the orthodromic population spike and the appearance of multiple population spikes in the CA1 pyramidal cell layer. d-Tubocurarine (4-100 microM), alpha-bungarotoxin (10-160 microM), and atropine (40-200 microM) had similar effects, although for alpha-bungarotoxin these excitatory effects were transient. Mecamylamine (1.6-3.2 mM) inhibited the population spike, while hexamethonium (3.2 mM) had no effect. These cholinergic antagonists were tested for their ability to block excitatory effects of nicotine (800 microM) at antagonist concentrations which were at or near threshold for intrinsic effects. Of the 5 antagonists tested, only mecamylamine (400 microM) effectively inhibited the nicotine-induced increase of the population spike amplitude and the appearance of multiple population spikes. These results suggest that nicotine exerts electrophysiological effects via a subclass of nicotinic cholinergic receptors that is neither neuromuscular nor ganglionic in the classical sense; these brain nicotinic receptors are sensitive to mecamylamine, but not to hexamethonium, alpha-bungarotoxin, or D-tubocurarine.

We examined the inbred mouse strains DBA/2Ibg, C57BL/6Ibg, and C3H/2Ibg for differences in susceptibility to bicuculline-induced seizures, as well as to bicuculline-induced epileptiform activity recorded in the CA1 pyramidal cell layer of hippocampal slices. For susceptibility to seizure onset the strain rank order was (most to least susceptible): C3H = DBA greater than C57. The rank order for sensitivity to effects of bicuculline on tonic seizure latency and on hippocampal epileptiform activity were identical: C3H greater than DBA = C57. It is suggested that mechanisms underlying the development of bicuculline-induced epileptiform events in the hippocampal slice may be similar to those involved in the development of tonic seizures measured in vivo.

Changes in plasma corticosterone (CCS) levels following intraperitoneal injections of nicotine were measured in four inbred mouse strains: DBA/2Ibg, C57BL/6Ibg, C3H/2Ibg, and A/J. In all four strains, nicotine produced a dose-dependent (0.5-2.0 mg/kg nicotine) increase in plasma CCS levels which peaked 10-30 min after injection. Saline increased plasma CCS levels in C57BL, A, and C3H, but not in DBA mice. After correcting for plasma CCS levels produced by saline injection, the nicotine-induced rise in plasma CCS was significantly lower for the C57BL strain than for the other three strains tested. These mouse strains also varied in their responses to saline injection with the rank order: C57BL greater than A = C3H greater than DBA. However, the two most divergent strains (C57BL and DBA) did not differ in the effects of a cold water stress. The response to nicotine was completely inhibited by mecamylamine in two strains tested (C3H and C57BL) whereas the response to saline injection was unaffected, suggesting that only the response to nicotine was mediated by nicotinic receptors. It is clear that elevations in plasma CCS induced either by saline injection or by nicotine are influenced by genetic factors.

Two analogues of the amino acid L-2-amino-4-phosphonobutanoic acid (L-APB) were synthesized in order to test the hypothesis that the dianionic nature of the side chain is responsible for antagonism of excitatory synapses in the hippocampal perforant path. These compounds, DL-2-amino-4-(methylphosphino)-butanoic acid (DL-AMPB) and O-methylphosphonyl-L-serine (O-MPLS), possess singly-charged side chains and yet display antagonistic activity, illustrating that a dianionic charge on the side chain is not necessary for antagonism. Comparing structure-activity relationships for DL-AMPB, O-MPLS, L-APB, and O-phospho-L-serine (O-PLS), patterns of synaptic activity emerged which suggest that substitution of a methyl group for one of the phosphoryl hydroxyl groups lowers ligand potency in both medial and lateral pathways. Also, the nature of the atom at the gamma-position appears to alter the potency and degree of pathway selectivity of these ligands, a methylene unit imparting more potency and selectivity than an oxygen atom.

The nitrogen excretory metabolism of the myxomycete Physarum polycephalum was studied. When cultured in partially defined broth medium or on agar, the principal excretory product was ammonia nitrogen. A small, variable quantity of urea was excreted in liquid culture. No uric acid or other purines were detected in the cultures. When microplasmodia were incubated with sodium [14C]bicarbonate, radioisotope was incorporated into citrulline, arginine, and urea. Incubation with L-[carbamoyl-14C]citrulline yielded labelled arginine, urea, and CO2. Substantial urease activity was found in extracts of the microplasmodia. These results, in conjunction with the lack of an absolute nutritional requirement for arginine, provide evidence that Physarum has a functional arginine biosynthetic pathway, an arginase, and a urease.

Four monomethyl-substituted analogues of 2-amino-4-phosphonobutanoic acid (APB), an antagonist of excitatory pathways in the central nervous system, were prepared in order to investigate the steric requirements of the APB receptor. Methyl groups were incorporated at the amino, alpha-, beta-, and gamma-positions. The beta- and gamma-methyl-substituted analogues of APB were found to be moderately potent antagonists in excitatory synapses of the hippocampal perforant path, as judged by extracellular recording techniques, while the N- and alpha-methyl-substituted analogues had much lower potencies. All of these APB analogues had very low potencies in the Schaffer collateral pathway. The APB receptors in the perforant path displayed more tolerance of methyl-substitution at the beta- and gamma-positions of APB than at the amino or alpha-positions in this system.

In addition to differing in ethanol sensitivity, long-sleep (LS) and short-sleep (SS) mice also differ in response to GABAergic agents. In the present study the sensitivity of LS and SS mice to the anesthetic, hypothermic and anticonvulsant effects of benzodiazepine, flurazepam, was determined. Flurazepam (75-300 mg/kg) induced a dose-dependent loss of righting response in both lines. The LS line displayed a two-fold greater sensitivity to the anesthetic effects of flurazepam. A dose-dependent decrease in body temperature was also observed following administration of flurazepam (25-150 mg/kg), but the two lines did not differ on this measure. Determination of the anticonvulsant effects of flurazepam (1-6 mg/kg) against seizures induced by 3-mercaptopropionic acid revealed that the SS line was more sensitive to the anticonvulsant effects of this benzodiazepine. These studies demonstrate that LS and SS mice differ in response to flurazepam, but the nature of the difference depends on the type of response measured and the dose of flurazepam employed.

Variable impairment occurs following birth associated brachial plexus injury. Muscle transpositions have evolved in response to patient need for functional reconstruction. Loss of external rotation limits the ability to perform activities with the arm in an overhead position. A modification of the combined latissimus dorsi and teres major transfer is described by means of which increased excursion of the transfer may be achieved.

Bath-application of nicotine (800 microM) to mouse hippocampal slices resulted in an increase in the amplitude of the population spike and the appearance of multiple population spikes in the CA1 pyramidal cell layer. Similar effects were observed after perfusion of the GABAA antagonist bicuculline methiodide (2 microM) and the glutamate decarboxylase inhibitor L-C-allylglycine (4 mM). These apparently excitatory effects of nicotine (800 microM) could be reversed by bath-application of gamma-aminobutyric acid (GABA; 400 microM), as well as by the GABA uptake inhibitor nipecotic acid (5 mM) and the benzodiazepine flurazepam (4 microM). Nicotine did not alter binding of [3H]GABA or [3H]flunitrazepam to whole brain plasma membranes. The results are consistent with the hypothesis that the electrophysiological effects of nicotine on CA1 pyramidal cell excitability is mediated by disruption of GABAergic transmission.

Eight kynurenic acid analogues were bath-applied to rat hippocampal slices while recording extracellular synaptic field potentials and the potencies of these analogues for inhibition of these responses were compared to that of kynurenic acid. Quinaldic acid, 4-hydroxyquinoline, 4-hydroxypicolinic acid, L-kynurenine and picolinic acid inhibited evoked field potentials, but were at least 15-fold less potent than kynurenic acid in all pathways tested. Xanthurenic acid was inactive in the pathways tested. Quinolinic acid and dipicolinic acid showed signs of agonist activity with IC50's of approx. 400 microM and 2500 microM, respectively. These studies show that the 2-carboxy group and the 4-hydroxy moiety are essential for the antagonist activity exhibited by kynurenate. They also show that the unsubstituted second aromatic ring greatly enhances the affinity of kynurenate for these receptors and that substitution in at least one position on this aromatic ring abolishes activity.

We previously demonstrated in the Schaffer collateral-CA1 region of the hippocampus that bath-applied agonists could be distinguished from antagonists among a group of acidic amino acid analogues by extracellular recording techniques. Here we report the use of the extracellular signs of agonist activity for discerning agonists and antagonists among several gamma-substituted glutamate analogues tested in the perforant path. The two-pathway composition of the perforant path offers the advantage over CA1 in that pathway-specificity, a postulated characteristic of antagonists, may be tested. By extracellular recording, D- and L-homocysteic acid, L-serine-O-sulfate, and L-2-amino-4-(5-tetrazolyl)-butanoic acid [L-glutamate tetrazole] were identified as agonists, and all 4 analogues were more potent than L-glutamate for inhibiting synaptic field potentials. Two previously identified antagonists, L-2-amino-4-phosphonobutyric acid and L-O-phosphoserine, exhibited the pathway-specificity and inhibitory kinetics consistent with properties expected for antagonists; both compounds detected 3 perforant path components with the same rank in sensitivity, suggesting that they are acting on the same set of receptors.

We used the hippocampal slice to examine extracellular electrophysiological responses to nicotine and the difference in sensitivity to nicotine-induced electrophysiological effects between the DBA and C3H mouse strains. Nicotine enhanced CA1 population spikes (PS) evoked by Schaffer collateral stimulation in a concentration-dependent manner (100 microM to 3.2 mM). This enhancement was slow to appear, achieving a maximum after 15 min. The enhanced PS was accompanied by the development of epileptiform activity (multiple PS's) at nicotine concentrations greater than or equal to 400 microM. Slices from DBA mice were significantly more sensitive than those from the C3H strain to these electrophysiological effects of nicotine. Mecamylamine (400 microM) blocked both the nicotine-induced PS enhancement and secondary population spikes in both strains, suggesting the involvement of nicotinic cholinergic receptors.