Voltage-sensitive dye imaging of transcranial magnetic stimulation-induced intracortical dynamics

Transcranial magnetic stimulation (TMS) is widely used in clinical interventions and basic neuroscience. Additionally, it has become a powerful tool to drive plastic changes in neuronal networks. However, highly resolved recordings of the immediate TMS effects have remained scarce, because existing recording techniques are limited in spatial or temporal resolution or are interfered with by the strong TMS-induced electric field. To circumvent these constraints, we performed optical imaging with voltage-sensitive dye (VSD) in an animal experimental setting using anaesthetized cats. The dye signals reflect gradual changes in the cells'' membrane potential across several square millimeters of cortical tissue, thus enabling direct visualization of TMS-induced neuronal population dynamics. After application of a single TMS pulse across visual cortex, brief focal activation was immediately followed by synchronous suppression of a large pool of neurons. With consecutive magnetic pulses (10 Hz), widespread activity within this “basin of suppression” increased stepwise to suprathreshold levels and spontaneous activity was enhanced. Visual stimulation after repetitive TMS revealed long-term potentiation of evoked activity. Furthermore, loss of the “deceleration–acceleration” notch during the rising phase of the response, as a signature of fast intracortical inhibition detectable with VSD imaging, indicated weakened inhibition as an important driving force of increasing cortical excitability. In summary, our data show that high-frequency TMS changes the balance between excitation and inhibition in favor of an excitatory cortical state. VSD imaging may thus be a promising technique to trace TMS-induced changes in excitability and resulting plastic processes across cortical maps with high spatial and temporal resolutions.Over recent decades, transcranial magnetic stimulation (1) (TMS) has become a frequently used method for noninvasive diagnostics, therapeutic treatment, and intervention for neurorehabilitation of neurological disorders (2–8). Additionally, TMS has proved a valuable tool in basic brain research as its perturbative effects allow area-selective manipulation of immediate cortical function (9–11), as well as its long-lasting alteration through plasticity and learning protocols (12, 13). However, direct measurements of the TMS-induced cortical dynamics at highly resolved spatiotemporal scales are missing because “online approaches” (14), using modern neuroimaging techniques such as functional MRI (fMRI) (15–18), magnetoencephalography (19), EEG (20), and near-infrared (21) or intrinsic optical imaging (22), are limited in either spatial or temporal resolutions or in both.Here we overcame these limitations, using optical imaging with voltage-sensitive dyes (VSD), which exploits the dye’s property to transduce gradual changes in voltage across neuronal membranes into fluorescent light signals. In contrast to imaging methods applicable in humans, this method is invasive but allows avoiding the commonly experienced contamination of signals by artifacts due to the strong TMS-induced electric field. In combination with a tandem-lens system of large numerical aperture (23) and a fast CCD camera as detector, VSD imaging captures several square millimeters of cortex with an emphasis on superficial layers (24–32), allowing us to record activity changes within milliseconds across millions of neurons at once with a spatial resolution of ∼50 μm (for review see ref. 33). We measured activity in cat primary visual cortex (V1) upon repetitive TMS (rTMS) (0.15 Hz, 1 Hz, and 10 Hz) and describe its effects on fundamental processing characteristics during subsequent visual stimulation.     

Neurophysiologic changes and bound calcium dynamics during the elaboration of associative learning in Helix lucorum]

Neurophysiological effects and dynamics of bound calcium (Ca-b) in command neurons of defense behavior (LP11 and RP11) during food aversion conditioning were studied in snail Helix lucorum. It was found that during associative learning both conditioned reaction and nonspecific facilitation of sensory responses were determined in neurons. The nonspecific facilitation of sensory responses was similar to that found during sensitization development in neurons. However, conditioned response appeared 30 min later than long-term sensitization development. Specific dynamics of the Ca-b level in neurons was obtained after three and more conditioning. The dynamics of the Ca-b level correlated with neurophysiological effects and differed from those measured during sensitization development. It is suggested that molecular mechanisms underlying associative learning and sensitization are different in a command neuron of defense behavior in the snail.     

Construction of SIV/HIV-1 chimeric viruses having the IL-5 gene and determination of their ability to replicate and produce IL-5

Summary.  During the progression of AIDS, there is a shift in abundance of immune cells from Th1-producing cells to Th2-producing cells. To determine whether this change might have an effect on HIV-1 replication in vivo, we constructed simian/human immunodeficiency chimeric viruses having the human IL-5 gene (a Th2-type cytokine) and examined the effect of the inserted gene on viral replication, IL-5 production and viral stability in vitro. The DNA of human IL-5 was inserted into vpr-deleted and nef-deleted infectious SHIVs. The obtained replication-competent viruses were used to infect human T-cell lines and monkey peripheral blood mononuclear cells. As a result, at the time of peak NI-IL5 virus production, IL-5 was produced with a significantly higher titer than 3sj-IL5. The functionality of the produced IL-5 was confirmed by IL-5-dependent cells. The replication of both SHIVs having IL-5 appeared to be faster than that of the parental viruses without the IL-5 gene. These results show that co-expression of IL-5 stimulates SHIV replication in vitro. Thus, it is expected that expression of IL-5 will also have an effect on viral replication and pathogenicity in vivo. Accepted February 8, 2001 Received October 17, 2000     

PCR diagnosis of sequences of a novel human herpes virus type 8 in patients with Kaposi sarcoma in Russia

Associations of a new human herpesvirus type 8 (HHV-8) with different forms of Kaposi's sarcoma (KS) in Russia have been studied. Search for this virus genetic information has been carried out in biopsy specimens of benign and malignant tumors other than KS, and probable sites of HHV-8 latency in human body have been checked. HHV-8 sequences were detected by polymerase chain reaction (PCR). HHV-8 sequences were most often detected in idiopathic (80.6%), AIDS-associated (80%), and immunosuppressive (100%) KS. The results indicate a selective association of HHV-8 with KS. No probable sites of the virus latency were detected in peripheral blood cells of patients with KS and in the prostate of patients with chronic prostatitis. The only exception was the husband of a patient with KS: HHV-8 sequences were detected in his prostatic secretion by nested PCR.     

Construction of an SIV/HIV type 1 chimeric virus with the human interleukin 6 gene and its production of interleukin 6 in monkey and human cells

The switch from a Th1- to a Th2-type cytokine response is reported to be involved in human immunodeficiency virus (HIV) disease progression. To study the effect of IL-6, one of the Th2-type cytokines, on AIDS pathogenesis, we constructed an SIV/HIV-1 chimeric virus (SHIV) having the human IL-6 gene (SHIV-IL6) SHIV-IL6 could replicate in M8166, a human T cell line, as well as in monkey and human peripheral blood mononuclear cells (PBMCs). Along with the SHIV-IL6 replication, IL-6 was detected in the culture supernatant by ELISA. The maximum level of IL-6 was 35, 15, and 8 ng/ml in M8166, human PBMCs, and monkey PBMCs, respectively. The expressed IL-6 was biologically active as shown by the proliferation of IL-6-dependent murine hybridoma (MH-60) cells. The inserted IL-6 gene was stable for at least four passages (45 days after the initial infection) in M8166 cells, suggesting the ability to achieve stable expression of IL-6 in long-term experiments. Therefore, we successfully established an SHIV system expressing IL-6, and this is the first report of an SHIV expressing a Th2-type cytokine. With this system, IL-6 should be expressed in the regions where the virus replicates, and therefore the inoculation of macaque monkeys with SHIV-IL6 is expected to provide further information on the etiology of AIDS.     

The Role of DNA Methylation and Activity of Neurotransmitter Receptors in the Mechanisms of Specific Anterograde Amnesia and Memory Recovery

Bulletin of Experimental Biology and Medicine - Impairment of reconsolidation of conditioned food aversion memory led to the development of a specific anterograde amnesia: repeated training of...     

The involvement of intracellular calcium in the sensitization of command neurons in the defensive behavior of the edible snail

Examinations carried out on command neurons of defensive behaviour of snail carried out using electrophysiological methods and a chlorotetracycline fluorescent probe have revealed that a single sensitizing action induces alterations of electrical neuronal activity and bound calcium (Ca-C) level in cells. An initial increase of the Ca-c amount (the first 15-20 min after the sensitizing action) coincides in time with depolarization, enhancement of plasma membrane excitability as well as with a decrease of amplitude and duration of the IPSPs induced by sensory stimulations. Repeated pronounced increase of the Ca-c level develops 50-60 min after the sensitizing action and correlates with facilitation of neuronal responses to sensory stimuli. Alteration of the Ca-c level in command neurons of defensive behaviour in the course of sensitization development differed from the previously described Ca-c shifts in the same cells in the course of habituation development in dynamics and direction.     

Effects of antisense oligonucleotides to mRNA for the early gene zif268 on the mechanisms of synapse-specific plasticity

Acquisition of nociceptive sensitization in common snails was accompanied by long-term facilitation of the responses of defensive behavior command neuron LPl1 to sensory stimulation of chemoreceptors on the head and mechanoreceptors on the head and foot. Acquisition of sensitization during intracellular administration of antisense oligonucleotides to mRNA encoding the early gene zif268 showed suppression of synaptic facilitation in the responses of neuron LPl1 to tactile and chemical stimulation of the snail’s head. Synaptic facilitation in the responses to tactile stimulation of the foot developed as in neurons of control sensitized animals. These results suggest that the early gene zif268 is selectively involved in the mechanisms of the specific regulation of the synaptic inputs of neuron LPl1 from sensory receptors on the snail’s head. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 56, No. 4, pp. 499–505, July–August, 2006.