Rebecca C. Klein

Multi-session transcranial direct current stimulation (tDCS) elicits inflammatory and regenerative processes in the rat brain.

Transcranial direct current stimulation (tDCS) is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types in vitro, but effects in vivo remain to be shown. Hypothesizing that tDCS might elicit the recruitment of cells to the cortex, we here studied the effects of tDCS in the rat brain in vivo. Adult Wistar rats (n = 16) were randomized to either anodal or cathodal stimulation for either 5 or 10 consecutive days (500 µA, 15 min). Bromodeoxyuridine (BrdU) was given systemically to label dividing cells throughout the experiment. Immunohistochemical analyses ex vivo included stainings for activated microglia and endogenous neural stem cells (NSC). Multi-session tDCS with the chosen parameters did not cause a cortical lesion. An innate immune response with early upregulation of Iba1-positive activated microglia occurred after both cathodal and anodal tDCS. The involvement of adaptive immunity as assessed by ICAM1-immunoreactivity was less pronounced. Most interestingly, only cathodal tDCS increased the number of endogenous NSC in the stimulated cortex. After 10 days of cathodal stimulation, proliferating NSC increased by ∼60%, with a significant effect of both polarity and number of tDCS sessions on the recruitment of NSC. We demonstrate a pro-inflammatory effect of both cathodal and anodal tDCS, and a polarity-specific migratory effect on endogenous NSC in vivo. Our data suggest that tDCS in human stroke patients might also elicit NSC activation and modulate neuroinflammation.

Progressive loss of synaptic integrity in human apolipoprotein E4 targeted replacement mice and attenuation by apolipoprotein E2.

Inheritance of the APOE4 allele is a well established genetic risk factor linked to the development of late onset Alzheimers disease. As the major lipid transport protein in the central nervous system, apolipoprotein (apo) E plays an important role in the assembly and maintenance of synaptic connections. Our previous work showed that 7 month old human apoE4 targeted replacement (TR) mice displayed significant synaptic deficits in the principal neurons of the lateral amygdala, a region that is critical for memory formation and also one of the primary regions affected in Alzheimers disease, compared to apoE3 TR mice. In the current study, we determined how age and varying APOE genotype affect synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in C57BL6, apoE knockout, and human apoE3, E4 and E2/4 TR mice at 1 month and 7 months. The apoE4 TR mice exhibited the lowest level of excitatory synaptic activity and dendritic arbor compared to other cohorts at both ages, and became progressively worse by 7 months. In contrast, the apoE3 TR mice exhibited the highest synaptic activity and dendritic arbor of all cohorts at both ages. C57BL6 mice displayed virtually identical synaptic activity to apoE3 TR mice at 1 month; however this activity decreased by 7 months. ApoE knockout mice exhibited a similar synaptic activity profile with apoE4 TR mice at 7 months. Consistent with previous reports that APOE2 confers protection, the apoE4-dependent deficits in excitatory activity were significantly attenuated in apoE2/4 TR mice at both ages. These findings demonstrate that expression of human apoE4 contributes to functional deficits in the amygdala very early in development and may be responsible for altering neuronal circuitry that eventually leads to cognitive and affective disorders later in life.

Functional somato‐dendritic α7‐containing nicotinic acetylcholine receptors in the rat basolateral amygdala complex

Multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed in the CNS. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. In the present study, whole‐cell patch‐clamp electrophysiological techniques were used to identify and characterize the presence of functional somato‐dendritic nAChRs within the basolateral complex of the amygdala. Pressure‐application of acetylcholine (ACh; 2 mm) evoked inward current responses in a subset of neurons from both the lateral (49%) and basolateral nuclei (72%). All responses displayed rapid activation kinetics, and were blocked by the α7‐selective antagonist methyllycaconitine. In addition, the α7‐selective agonist choline induced inward current responses that were similar to ACh‐evoked responses. Spiking patterns were consistent with pyramidal class I neurons (the major neuronal type in the basolateral complex); however, there was no correlation between firing frequency and the response to ACh. The local photolysis of caged carbachol demonstrated that the functional expression of nAChRs is located both on the soma and dendrites. This is the first report demonstrating the presence of functional nAChR‐mediated current responses from rat amygdala slices, where they may be playing a significant role in fear and aversively motivated memory.

Inhibition of nicotinic acetylcholine receptors by apolipoprotein E-derived peptides in rat hippocampal slices

Apolipoprotein E (ApoE) is a well-known genetic risk factor for Alzheimers disease (AD). Dysfunctions in cholinergic signaling, and in particular in the function of neuronal nicotinic acetylcholine receptors (nAChRs), have also been linked with AD and cognition. To address whether there is a link between ApoE and nAChR function, we used electrophysiological techniques to test the effects of synthetic ApoE-mimetic peptides derived from the low-density lipoprotein receptor (LDLR) binding domain for the ability to modulate nAChR activity in hippocampal interneurons. ApoE(133-149) completely inhibited ACh-evoked responses in a dose-dependent manner, yielding an IC(50) value of 720+/-70 nM. A shorter peptide spanning residues 141-148 mimicked this effect while a second peptide spanning residues 133-140 was without effect, indicating that the arginine-rich domain is responsible for nAChR interaction. Inhibition of ACh-evoked responses was voltage-independent, and displayed partial receptor specificity as no effect on glycine- or GABA-evoked responses occurred. These results demonstrate that peptides derived from the LDLR binding domain of ApoE block the function of nAChRs in hippocampal slices, an interaction that may have implications for AD.

Adolescent intermittent alcohol exposure: persistence of structural and functional hippocampal abnormalities into adulthood.

BACKGROUND Human adolescence is a crucial stage of neurological development during which ethanol (EtOH) consumption is often at its highest. Alcohol abuse during adolescence may render individuals at heightened risk for subsequent alcohol abuse disorders, cognitive dysfunction, or other neurological impairments by irreversibly altering long-term brain function. To test this possibility, we modeled adolescent alcohol abuse (i.e., intermittent EtOH exposure during adolescence [AIE]) in rats to determine whether adolescent exposure to alcohol leads to long-term structural and functional changes that are manifested in adult neuronal circuitry. METHODS We specifically focused on hippocampal area CA1, a brain region associated with learning and memory. Using electrophysiological, immunohistochemical, and neuroanatomical approaches, we measured post-AIE changes in synaptic plasticity, dendritic spine morphology, and synaptic structure in adulthood. RESULTS We found that AIE-pretreated adult rats manifest robust long-term potentiation, induced at stimulus intensities lower than those required in controls, suggesting a state of enhanced synaptic plasticity. Moreover, AIE resulted in an increased number of dendritic spines with characteristics typical of immaturity. Immunohistochemistry-based analysis of synaptic structures indicated a significant decrease in the number of co-localized pre- and postsynaptic puncta. This decrease is driven by an overall decrease in 2 postsynaptic density proteins, PSD-95 and SAP102. CONCLUSIONS Taken together, these findings reveal that repeated alcohol exposure during adolescence results in enduring structural and functional abnormalities in the hippocampus. These synaptic changes in the hippocampal circuits may help to explain learning-related behavioral changes in adult animals preexposed to AIE.

Paired‐pulse potentiation of α7‐containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones

Diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including fast‐desensitizing α7‐containing receptors, are expressed in the CNS. While nAChRs appear to regulate cognitive processing and synaptic plasticity, little is known to date about how this regulation occurs, particularly in brain regions known to be important for cognition. By combining patch‐clamp electrophysiology with local photolysis of caged carbachol to rapidly activate the α7‐containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices, we describe a novel transient up‐regulation of channel function. The nAChRs were activated using a paired‐pulse uncaging protocol, where the duration of the UV laser pulses (5–25 ms) and the interval between pulses (200 ms to 30 s) were varied. At relatively long interpulse intervals, we observed a strong (> 75%) decrease in the amplitude of the second response due to desensitization. However, when two pulses were applied at a 200 ms interval, a > 3‐fold increase in the amplitude of the second response was observed, a phenomenon referred to here as paired‐pulse potentiation. Interestingly, this potentiation appeared to be regulated by [Ca2+]i, and/or Ca2+‐dependent processes, as it was significantly enhanced by dialysing cells with either the Ca2+ chelator BAPTA, or with peptide inhibitors of either calcineurin or PKC, and was attenuated by dialysing cells with the CaMKII inhibitor KN‐93. No potentiation was observed using caged GABA or glutamate, indicating some specificity for nAChRs. Thus, rat hippocampal α7‐containing nAChRs possess a newly described phenomenon of paired‐pulse potentiation that may be involved in regulating synaptic plasticity in the hippocampus.

Inhibition of NMDA-induced currents by conantokin-G and conantokin-T in cultured embryonic murine hippocampal neurons

Conantokin-G (con-G) and conantokin-T (con-T) are small (17 and 21 amino acids, respectively) gamma-carboxyglutamate (Gla) containing peptides derived from the venoms of marine cone snails that are potent and selective inhibitors of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of con-G and con-T on NMDA-evoked responses were evaluated in mouse primary hippocampal neuronal cultures using the whole-cell patch-clamp technique. Under equilibrium conditions, NMDA-induced currents were inhibited by con-G and con-T (10 nM-100 microM) in a dose-dependent manner while maintaining a holding potential of -70 mV. In the presence of saturating amounts of NMDA (100 microM) and glycine (1 microM), the IC50 values obtained were 487 +/- 85 nM for con-G and 1030 +/- 130 nM for con-T. NMDA (10 microM-1 mM) dose-response curves produced in the presence of con-G or con-T (1 or 3 microM) resulted in a downward shift of the current response at saturation with NMDA, without affecting the EC50. The maximum response obtainable in the absence of peptide could not be achieved by increasing concentrations of NMDA. The same effect was also observed for conantokin inhibition of spermine-potentiated responses. Association rate constants (k(on)) for the peptides were determined in the presence of NMDA and glycine, with and without the addition of spermine. Using a single binding site bimolecular model, k(on) values were 3.1 +/- 0.2 x 10(3) M(-1) s(-1) for con-G and 3.2 +/- 0.1 x 10(3) M(-1) s(-1) for con-T in the absence of spermine. The added presence of a saturating amount of spermine (300 microM) resulted in an approximate 60% increase in the k(on) values for both con-G and con-T. These results demonstrate that con-T and con-G inhibit NMDA-evoked currents, as well as the potentiation by spermine, in what appears to be a noncompetitive manner, and that spermine increases the rate of conantokin inhibition.

Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides.

The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] displayed 5.6- and 8.4-fold decreases in tau(off), respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.

Inhibition of MK801 binding in adult rat brain sections by conantokin-G and conantokin-T.

The functional interactions of conantokins with anatomical sites in rat brain have been assessed through displacement of the non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, dizocilpine (MK801). The binding of (+)-3-[125I]-iodo-MK801 (1 nM) to coronal sections from adult rat brain was inhibited in a dose-dependent manner by conantokin-T (con-T) and conantokin-G (con-G). Quantitative densitometry was used to determine IC50 values for conantokin inhibition of [125I]-MK801 binding in the cortex, thalamus and hippocampus. Con-T completely inhibited [125I]-MK801 specific binding in all brain regions at a saturating concentration of 100 microM. Con-G was able to completely displace [125I]-MK801 in the cortex and thalamus, but only inhibited this same binding up to approximately 90% in the hippocampus. Both peptides maintained their inhibitory properties in the presence of 1 mM EDTA, suggesting that divalent cations are not required for their action in this regard. The added presence of spermine (150 microM) resulted in a two-fold increase in [125I]-MK801 binding and a two-fold decrease in the IC50 values for both peptides. The data obtained in this investigation further demonstrate that [125I]-MK801 is a useful probe for the indirect determination of functional NMDAR ligand binding sites in rat brain sections.

Amino acid determinants for NMDA receptor inhibition by conantokin‐T

Several derivatives of conantokin‐T (con‐T), a naturally occurring, γ‐carboxyglutamate (Gla)‐containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [3H]MK‐801 from adult rat forebrain membranes. Analyses of progressive C‐terminal truncation analogs of the parent 21‐mer revealed gradual losses in activity with decreased chain length. In this series, con‐T[1–8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side‐chain length and charge in the N‐terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism‐derived helical contents of the con‐T derivatives. Further elaboration of structure‐function relationships in con‐T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con‐G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C‐terminal regions. Chimeras of con‐T and con‐G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin‐based NMDAR inhibitors.