Functional characteristics of Schaffer's collaterals studied in vivo and in vitro

A comparative analysis was made of the functional characteristics of connections between hippocampal areas CA₃ and CA₁ (Schaffer's collaterals) in experiments in vivo on unanesthetized rabbits and in vitro on surviving slices of guinea pig hippocampus, with extracellular recording in area CA₁. In the case of electrical stimulation of the collaterals in vitro, post-activational inhibition was weak, responses of inhibition of spontaneous discharges were absent, and low frequencies of stimulation were more effective than in vivo. Posttetanic changes were found more frequently in experiments in vitro and they lasted longer than in vivo. The predominant effect of tetanization under normal conditions was depression, but during incubation it was facilitation of responses. The possible causes of these differences are discussed.Institute of Biophysics, Academy of Sciences of the USSR, Puschino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 3, pp. 208–217, May–June, 1979.     

Effect of stimulation of neocortical regions and the subiculum on hippocampal neurons in rabbits

In chronic experiments on rabbits using extracellular recording of unit activity in hippocampal area CA₁ the effects of stimulation of the subiculum, posterior cingulate cortex, and anterior and posterior nonprimary areas of the neocortex were investigated. The effects of such stimulation were compared in the intact and chronically isolated hippocampus. It is concluded from the results that direct two-way connections exist between CA₁ and the subiculum. Polysynaptic influences of the subiculum on CA₁ are realized through the entorhinal cortex, for they are not present in the isolated hippocampus. Influences of the neocortical areas studied on CA₁ are transmitted to some extent through a relay in the subiculum. The entorhinal cortex plays no part in the realization of polysynaptic effects. The effectiveness of these influences increases with removal of the principal hippocampal afferent systems.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 315–323, May–June, 1982.     

Comparison of direct influences of the perforant path on hippocampal CA1 and CA3 neurons in vitro

The efficiency of synapses of the perforant path located on terminals of apical dendrites of CA₁ and CA₃ neurons was investigated in sections of the guinea pig hippocampus in vitro. Neurons of both areas were shown to respond to stimulation of the perforant path by action potential generation. Responses of most CA₁ neurons appeared to repetitive stimulation with a frequency of up to 30–80/sec. Neurons in area CA₃ respond only to low-frequency stimulation (under 5/sec). Posttetanic potentiation of responses to stimulation of the perforant path was found in both areas of the hippocampus.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 4, pp. 303–310, July–August, 1979.     

Effect of electrical stimulation of the septum on subicular neurons with different kinds of spontaneous activity in rabbits

The effect of electrical stimulation of the medial nucleus of the septum and of hippocampal area CA₁ on subicular neurons with three different types of spontaneous activity (with theta-modulation, with delta-modulation and complex spikes, and with irregular single-spike activity) was studied in unanesthetized rabbits by extracellular recording of unit activity. Cells with theta-activity were found to respond in a distinctive functional manner to stimulation of the medial nucleus of the septum: Their reactivity was higher but latent periods of their responses were much shorter than those of cells with delta-activity and irregular discharges. Stability of modulation of theta-cell activity increased both during and after stimulation of the medial septal nucleus. Electrical stimulation of hippocampal area CA₁, on the other hand, led to disappearance of rhythm modulation. Average response latencies of all three types of cells to stimulation of area CA₁ were equal. The results indicate special properties of the septal input to subicular cells with theta-modulation.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 326–333, May–June, 1985.     

Investigation of heterosynaptic interactions in hippocampal areas CA1, and CA3 in vitro

Heterosynaptic interactions between synapses located at a considerable distance from the cell body (perforant path) and lying close to the body of the neuron (synapses of Schaffer's collaterals and axons of the dentate fascia) on guinea pig hippocampal neurons were investigatedin vitro. It was shown by the paired stimulus method that, using stimulation of subthreshold intensity for action potential generation, spatiotemporal summation takes place in both pairs of synaptic systems. If above-threshold stimulation was used, afferents lying close to the cell body suppressed responses evoked by stimulation of distant afferents for a longer time (up to 20 msec in area CA₁ and up to 300 msec in area CA₃) than during the opposite combination of stimuli (up to 3–8 msec). After tetanization of the dentate fascia depression of responses of area CA₃ neurons to stimulation of the perforant path was observed for 2–30 min. In the remaining cases, no significant prolonged heterosynaptic posttetanic changes were observed. The possible mechanisms of these interactions are discussed.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 524–532, November–December, 1979.     

Frequency potentiation in the neonatal rabbit hippocampus

The formation of properties of frequency potentiation in the entorhinal afferent pathway of the hippocampus was studied in unanesthetized rabbits aged from 1 to 15 days. In areas CA₁ and CA₃ of the dorsal hippocampus in newborn rabbits repetitive (1–20 Hz) electrical stimulation of the perforant path led to an increase in amplitude of the slow wave of the field potential by 20–100% compared with the control and to an increase in the probability of response discharges from the neurons from 0–0.5 in the control to 0.8–1.0 during tetanization. In rabbits aged 2–3 days potentiation was more marked at a frequency of 4–6 Hz, whereas depression of the responses developed rapidly to a higher frequency of stimulation. The frequency optimum of 4–15 Hz was established on the 5th day. Potentiation of the first component of the field potential was observed starting from the 8th–10th day of life. The experimental results show that the property of frequency potentiation in the cortical afferent connections of the hippocampus is found in rabbits actually at birth, and it acquires the adult form at the beginning of the second week of life.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 533–539, November–December, 1979.     

Effect of reticular formation on hippocampal neurons (field CA1)

The reaction of field CA₁ hippocampal neurons to stimulation of the reticular formation (RF) with impulses of different frequencies was investigated in experiments on unanesthetized rabbits. The effect of electrical and sensory stimuli was compared and the effect of reticular stimulation on the sensory responses was determined. With an increase in the frequency of RF stimulation, the number of neurons of field CA₁ responding with inhibition of the activity increases. Multimodal neurons of the hippocampus depend on the reticular input to a greater degree than unimodal neurons. Neurons whose activity does not change in response to the effect of sensory stimuli also do not respond to stimulation of the RF. Neurons responding with inhibitory reactions to sensory stimulation show a higher correlation with the effects of RF stimulation than neurons with activation reactions and, especially those with "complex" responses to the effect of sensory stimuli. In a considerable number of hippocampal neurons the responses to sensory stimuli change in the course of 10–15 min after stimulation of the RF. The role of the RF in the organization of the reactions of hippocampal neurons is discussed.Division of Memory Problems, Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oke. Translated from Neirofiziologiya, Vol. 3, No. 3, pp. 227–235, May–June, 1971.     

Posttetanic changes in hippocampal minimal evoked potentials

Changes in the EEG induced by a single spike were recorded in the hippocampus of an unanesthetized rabbit. Summation of focal electrical activity synchronous with spontaneous single unit discharges at the symmetrical point of contralateral hemisphere revealed no stable potentials which could reflect these changes. In two cases discharges identified as activity of Shaffer's collaterals were recorded in area CA₁. Summation of post-spike changes in evoked activity recorded by the same microelectrode showed stable negative waves with an amplitute of 40–60 µV, which could have been evoked by single spikes. The curve of amplitude of the averaged evoked potentials versus near-threshold current strength stimulating the intrahippocampal pathways was not smooth in most experiments but stepwise in character. It is suggested that the minimal evoked potential corresponding to the first step (amplitude 40–80 µV) reflects a response to stimulation of one fiber. After above-threshold tetanization prolonged posttetanic potentiation of the minimal evoked potentials did not arise in CA₁ in response to stimulation of Shaffer's collaterals. Minimal evoked potentials recorded in area CA₃ in response to stimulation of the dentate fascia showed clear potentiation. The results are in agreement with the hypothesis of the synaptic localization of the mechanisms responsible for prolonged posttetanic potentiation.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 124–134, March–April, 1977.     

Spatial distribution of hippocampal response to tooth pulp stimulation and acoustic stimulation

Evoked potentials (EP) and neuronal responses produced by tooth pulp stimulation and a clicking sound were recorded at different hippocampal sites using microelectrodes in unrestrained rats. Spatial distribution of EP was found to be the same for both types of stimulation. Averaged EP consisted of a high amplitude negative preceded by a low-amplitude positive component (N₁ and P₁, respectively). Latency of the N₁ wave reached its minimum (of 27 msec) at the middle third of the molecular layer of the dentate gyrus and the outer portion of the CA₃ apical dendrites. Latency of N₁ was considerably longer in the stratum radiatum layer of the CA₁. Laminar profiles of the amplitude of the N₁ componenent of EP produced in the dentate gyrus and the CA₃ by tooth pulp stimulation resemble those observed during perforant path stimulation; in the CA₁ they are similar to those evoked by stimulating the Schaffer collaterals. Maximum amplitude of the P₁ component was observed above the pyramidal layer of the CA₁ and the hilus. Neuronal discharge pattern changed in all hippocampal regions under the effects of both tooth pulp stimulation and the clicking sound. It is deduced that information can reach the hippocampus by two routes: via a "fast" (inhibitory) pathway through the fimbria and the fornix and a slower (excitatory) path through the entorhinal cortex.P. Flexig Institute for Brain Research, Karl Marx University, Leipzig, DR. Institute of Physiology, Pecs University Medical School, Pecs, Hungary. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 36–46, January–February, 1987.     

Sources of direct afferents of the rostral field CA3 in the rat dorsal hippocampus

Afferents to the rostral field CA₃ of the dorsal hippocampus were investigated using horseradish peroxidase retrograde transport techniques. By iontophoretic injection of horseradish peroxidase into this area of the hippocampus cells stained with this enzyme could be identified in the anterior nuclei of the thalamus, the supramillary and submamillothalamic nuclei of the hypothalamus, and the midbrain central gray matter, as well as the parietal, insular, temporal, retrosplenial, and pyriform areas of the neocortex. The findings obtained complete the picture of connections between one of the least explored sections of the rat hippocampus and other brain structures.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 469–475, July–August, 1986.     

Effect of noradrenalin and serotonin on responses of area CA3 neurons evoked in guinea pig hippocampal slices by electrical stimulation of mossy fibers

The effect of noradrenalin (NA) and serotonin (5-HT) on responses of area CA₃ cells evoked by electrical stimulation of mossy fibers was studied in slices of guinea pig hippocampus survivingin vitro. Both substances, which modify the general level and organization of spontaneous activity, also affected responses of area CA₃ cells. Changes in magnitude and structure of the response usually correlated with corresponding changes in spontaneous activity. In certain cases NA, which lowered the frequency of spontaneous activity but increased its relative content of "complex discharges" and also the number of reduced action potentials in the complex discharge, also led to an increase in the response to stimulation. 5-HT evoked periodic grouped activity in some cells and led to the appearance of such grouped discharges for the first time in the responses of other cells. Unlike NA, 5-HT caused prolonged (up to 40 min) after-facilitation of the response and an increase in spontaneous discharge frequency.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 410–417, July–August, 1982.     

Direct connections of the hippocampus with the retrosplenial cortex in rats

Connections of the retrosplenial cortex with the hippocampus in rats were investigated by the method of retrograde axonal transport of horseradish peroxidase (HRP). If a crystal of HRP was introduced into the dorsocaudal part of the retrosplenial cortex, HRP-labeled cells were found in the rostral pole of hippocampal area CA₃ and in the presubiculum. After iontophoretic injection of HRP into the rostral pole of the hippocampus, HRP-labeled cells were found in layers V and VI of the retrosplenial cortex and in the presubiculum. The results are evidence of reciprocity of direct connections between the rostral pole of the hippocampus and the retrosplenial cortex, and also of the existence of direct efferents of the dorsal subiculum to the rostral pole of the hippocampus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 102–107, January–February, 1985.     

Excitatory postsynaptic potentials of hippocampal neurons and their extinction during repeated stimulation

A method of detecting "minimal" excitatory postsynaptic potentials (EPSP) in neurons of hippocampal area CA₃ of the unanesthetized rabbit during stimulation of the septo-fimbrial region and the dentate fascia is described. The method consists of presenting a strong (a current of up to 1 mA) conditioning stimulus, inducing a distinct inhibitory postsynaptic potential (IPSP), before a near-threshold (current of 0.03–0.35 mA) testing stimulus. The response to the testing stimulus, develoing after the previous conditioning IPSP, in most cases was purely depolarizing and, judging from the change in the latent period in some cases and the absence of correlation between its amplitude and that of the IPSP, it is a pure EPSP. If the testing stimuli are presented at low enough frequency (intervals of not less than 1 sec) the amplitude of the EPSP evoked by them gradually falls. This decrease exhibits some of the characteristic properties of extinction of behavioral responses (recovery after an interruption, a more rapid decrease during repeated series of stimuli, a slower decrease in amplitude during less frequent stimulation). The amplitude of the IPSP also fell or showed no significant change. The results are evidence in support of the hypothesis that extinction is based on a mechanism of homosynaptic depression.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 10, No. 1, pp. 3–12, January–February, 1978.     

Functional development of hippocampal mossy fiber synapses in rabbits

The course of functional maturation with age of mossy fiber synapses on pyramidal cells in areas CA_3,4 of the dorsal hippocampus was investigated by extracellular recording of focal potentials and single unit responses of the hippocampus to electrical stimulation of the dentate fascia in waking, unimmobilized rabbits aged from 1 to 14 days. After the 4th day of postnatal life focal potentials appeared in response to single stimulation, in the form of a biphasic short-latency wave, characteristic of responses of the mature hippocampus, accompanied by spike discharges with a latent period of 3 to 10 msec and inhibitory responses of the hippocampal neurons. During the next 10 days the amplitude of the focal potentials increased from several hundred millivolts, with the sharpest increase observed from the 4th through the 7th days. In early age periods global and unitary responses were shown to be capable of frequency potentiation and also of short-term after-potentiation.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 3, pp. 246–254, May–June, 1980.     

Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (μ), delta (δ) and kappa (κ) opioid and nociceptin (NOP) receptors. Binding assays showed that 4–10 had subnanomolar K_i values for μ and κ opioid receptors. Functional assays for stimulation of [³⁵S]GTPγS binding showed that several compounds acted as partial or inverse agonists and antagonists of the μ and δ, κ opioid or NOP receptors. The compounds showed considerable stability in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the functional activity of human cytochrome P450s was examined to determine any potential inhibition by 4–9. Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a representative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, utilizing operant techniques showed 5–8 to have very potent efficacy (ED₅₀ values 19–50 μg/kg).     

Mechanisms of Left-Right Coordination in Mammalian Locomotor Pattern Generation Circuits: A Mathematical Modeling View

The locomotor gait in limbed animals is defined by the left-right leg coordination and locomotor speed. Coordination between left and right neural activities in the spinal cord controlling left and right legs is provided by commissural interneurons (CINs). Several CIN types have been genetically identified, including the excitatory V3 and excitatory and inhibitory V0 types. Recent studies demonstrated that genetic elimination of all V0 CINs caused switching from a normal left-right alternating activity to a left-right synchronized “hopping” pattern. Furthermore, ablation of only the inhibitory V0 CINs (V0_D subtype) resulted in a lack of left-right alternation at low locomotor frequencies and retaining this alternation at high frequencies, whereas selective ablation of the excitatory V0 neurons (V0_V subtype) maintained the left–right alternation at low frequencies and switched to a hopping pattern at high frequencies. To analyze these findings, we developed a simplified mathematical model of neural circuits consisting of four pacemaker neurons representing left and right, flexor and extensor rhythm-generating centers interacting via commissural pathways representing V3, V0_D, and V0_V CINs. The locomotor frequency was controlled by a parameter defining the excitation of neurons and commissural pathways mimicking the effects of N-methyl-D-aspartate on locomotor frequency in isolated rodent spinal cord preparations. The model demonstrated a typical left-right alternating pattern under control conditions, switching to a hopping activity at any frequency after removing both V0 connections, a synchronized pattern at low frequencies with alternation at high frequencies after removing only V0_D connections, and an alternating pattern at low frequencies with hopping at high frequencies after removing only V0_V connections. We used bifurcation theory and fast-slow decomposition methods to analyze network behavior in the above regimes and transitions between them. The model reproduced, and suggested explanation for, a series of experimental phenomena and generated predictions available for experimental testing.     

Responses of upper cervical spinal neurons in cats to stimulation of the brain-stem locomotor region at different frequencies

Synaptic responses of neurons in segments C₂ and C₃ to stimulation of locomotor points in the medulla or midbrain were recorded extracellularly in mesencephalic cats. Neurons generating responses with an index of 0.4–0.6 to stimulation with a frequency of 2 Hz maintained this same index at frequencies of 20–60 Hz. The discharge index of many neurons during stimulation at 2 Hz was low, and it increased to 0.4–0.6 when high-frequency stimulation was used. More than half of the cells were excited by stimulation of both ipsilateral and contralateral locomotor points; one-quarter of the neurons responded to stimulation of locomotor points in both medulla and midbrain. The cells studied were located 1.8–4.2 mm from the dorsal surface of the spinal cord. The mean latencies of responses with an index of not less than 0.5 lay within the range 2–30 msec, with a mode of 2–8 msec. Considerable fluctuations of latent period were observed for long-latency responses. The possibility that the neurons studied may participate in the transmission of activity from the locomotor region of the brain stem to stepping generators in the spinal cord is discussed.Institute for Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 15, No. 4, pp. 355–361, July–August, 1983.     

Functional properties of interneurons of the masticatory reflex

Interneurons of the supratrigeminal nucleus, transmitting effects from the sensory and motor branches of the trigeminal nerve to motoneurons of the muscles of mastication were investigated. Two groups of interneurons with different functional connections were found. The first group (A) contains neurons excited during stimulation of the sensory branches and the motor nerve to the digastric muscle (A₁), neurons excited during stimulation of sensory branches and high-threshold afferents of the motor nerve to the masseter muscle (A₂), and neurons excited only by low-threshold afferents of the motor nerve to the masseter muscle (A₃). Neurons of the second group (B) were activated only by sensory fibers of the trigeminal nerve. It is postulated that interneurons of group A transmit inhibitory effects to motoneurons of antagonist muscles of the lower jaw. Group B interneurons participate in the transmission of excitatory influences to motoneurons of the digastric muscle.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 2, pp. 150–157, March–April, 1972.     

Flow threshold for enhanced phorbol ester binding in the ischemic gerbil brain

The correlation between regional phorbol ester binding and cerebral blood flow (CBF) was evaluated in the gerbil brain after 2-hour unilateral common carotid artery occlusion [³H]phorbol 12, 13-dibutyrate (PDBu) was used as a specific ligand for estimating the translocation of protein kinase C (PKC), and CBF was determined by the [¹⁴C]iodoantipyrine method. A quantitative autoradiographic method permitted concurrent measurement of these two parameters in the same brain. In the ischemia group of the animals, statistically significant, inverse correlations were noted between the CBF and PDBu binding in the hippocampus (CA₁ and CA₃ regions and dentate gyrus), the caudate-putamen and lateral nuclei of the thalamus. In these regions, the PDBu binding increased progressively as CBF fell below 35–40 ml/100 g/min. On the other hand, the PDBu binding in the cerebral cortices did not show any significant changes even when CBF was decreased to below 35 ml/100 g/min. The above data suggest that (1) the translocation of PKC to the cell membrane may be regionally specific in response to ischmia and may remain in the regions particularly vulnerable to ischemia such as the hippocampus, caudate-putamen and lateral nuclei of the thalamus in the early ischemic phase; (2) the threshold of CBF below which PKC begins to translocate to the cell membrane in the above regions, may be 35–40 ml/100 g/min in 2-hour ischemia.     

Disruption of postnatal folliculogenesis and development of ovarian tumor in a mouse model with aberrant transforming growth factor beta signaling

Background

Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

Methods

A mouse model containing constitutively active TGFBR1 (TGFBR1^CA) using Gdf9-iCre (termed TGFBR1-CA^G9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CA^G9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3’-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues.

Results

TGFBR1-CA^G9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CA^G9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

Conclusions

These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.